US4603209A - Fluorescent indicator dyes for calcium ions - Google Patents
Fluorescent indicator dyes for calcium ions Download PDFInfo
- Publication number
- US4603209A US4603209A US06/648,649 US64864984A US4603209A US 4603209 A US4603209 A US 4603209A US 64864984 A US64864984 A US 64864984A US 4603209 A US4603209 A US 4603209A
- Authority
- US
- United States
- Prior art keywords
- compound
- ring
- esters
- amino
- pharmaceutically acceptable
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Lifetime
Links
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D307/00—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom
- C07D307/77—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom ortho- or peri-condensed with carbocyclic rings or ring systems
- C07D307/78—Benzo [b] furans; Hydrogenated benzo [b] furans
- C07D307/79—Benzo [b] furans; Hydrogenated benzo [b] furans with only hydrogen atoms, hydrocarbon or substituted hydrocarbon radicals, directly attached to carbon atoms of the hetero ring
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P5/00—Drugs for disorders of the endocrine system
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D209/00—Heterocyclic compounds containing five-membered rings, condensed with other rings, with one nitrogen atom as the only ring hetero atom
- C07D209/02—Heterocyclic compounds containing five-membered rings, condensed with other rings, with one nitrogen atom as the only ring hetero atom condensed with one carbocyclic ring
- C07D209/04—Indoles; Hydrogenated indoles
- C07D209/08—Indoles; Hydrogenated indoles with only hydrogen atoms or radicals containing only hydrogen and carbon atoms, directly attached to carbon atoms of the hetero ring
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D215/00—Heterocyclic compounds containing quinoline or hydrogenated quinoline ring systems
- C07D215/02—Heterocyclic compounds containing quinoline or hydrogenated quinoline ring systems having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen atoms or carbon atoms directly attached to the ring nitrogen atom
- C07D215/16—Heterocyclic compounds containing quinoline or hydrogenated quinoline ring systems having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen atoms or carbon atoms directly attached to the ring nitrogen atom with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
- C07D215/38—Nitrogen atoms
- C07D215/40—Nitrogen atoms attached in position 8
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D413/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and oxygen atoms as the only ring hetero atoms
- C07D413/02—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and oxygen atoms as the only ring hetero atoms containing two hetero rings
- C07D413/04—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and oxygen atoms as the only ring hetero atoms containing two hetero rings directly linked by a ring-member-to-ring-member bond
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
- Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10S436/00—Chemistry: analytical and immunological testing
- Y10S436/80—Fluorescent dyes, e.g. rhodamine
Definitions
- the present invention relates to fluorescent indicator dyes that are specific for calcium ions. More specifically, the present invention relates to fluorescent, calcium-specific indicator dyes that fluoresce at different wavelengths depending on whether dye has bound calcium or is free of calcium. This wavelength shift makes it possible to measure calcium concentrations on an absolute scale by simply determining the ratio of fluorescence at the two wavelengths.
- the present invention relates to an improved method for synthesizing alpha-acyloxyalkyl bromides, especially acetoxymethyl bromide which is useful in preparing the acetoxymethyl esters contained within the class of new fluorescent dyes disclosed herein.
- Cytosolic esterases then split off the ester groups, leaving the membrane-impermeant quin2 tetra-anion trapped in the cytosol.
- the fluorescence of quin2 provides a measure of cytosolic free calcium concentrations. It is calibrated by interpolation between the limiting fluorescence levels obtained by forcing the trapped dye to known saturatingly high as well as vanishingly low levels of calcium.
- quin2 Although quin2 has revealed much important biological information, it has severe and acknowledged limitations (see references 5 and 7). For example, quin2's preferred excitation wavelength of 339 nm is too short since it falls within the ultraviolet range. UV irradiation excites significant autofluorescence from the cells being monitored. In addition, such UV irradiation could cause biological side effects in the cells being tested. Also, it is known that light in this UV range does not penetrate microscope optics very well.
- a second problem with quin2 is the fact that its extinction coefficient ( ⁇ 5000) and fluorescence quantum yield (0.03 to 0.14), though comparable to dansyl groups in aqueous solution, are too low. These spectroscopic properties mean that quin2 loadings of several tenths of millimolar or more are necessary to overcome cell autofluorescence. In some cell types, this much loading significantly buffers [Ca 2+ ] i transients.
- a third problem with quin2 is the fact that it signals Ca 2+ by increasing its fluorescence intensity without much shift in either excitation or emission wavelengths.
- fluorescence intensity is also dependent on other poorly quantified or variable factors such as illumination intensity, emission collection efficiency, dye concentration, and effective cell thickness in the optical beam.
- an indicator dye that responded to calcium by shifting wavelengths while still maintaining strong fluorescence.
- the ratio of the fluorescences of two suitably chosen wavelengths would then signal calcium while cancelling out most or all of the possible variability due to instrument efficiency and content of effective dye.
- a fourth problem with quin2 is that it does not measure calcium at micromolar levels.
- the high effective affinity of quin2 for Ca 2+ is ideal for measuring levels near or below typical resting values of [Ca 2+ ] i , i.e., near 10 -7 M.
- quin2 approaches saturation so that further changes in fluorescence intensity are small.
- dyes with weaker affinities are needed to quantify calcium at these elevated levels.
- the new fluorescent indicator compounds should exhibit a wavelength shift upon Ca 2+ -binding.
- the new compounds should also overcome some of the specific problems associated with use of the known Ca-specific indicator, quin2. More specifically, as compared to quin 2, the new compounds should show much stronger fluorescence, a somewhat weaker affinity for Ca 2+ and a better selectivity against magnesium and heavy metals.
- FIG. 1 is a chemical flow chart illustrating the synthetic pathway leading to stil-1, stil-2, and indo-1.
- FIG. 2 is a chemical flow chart illustrating the synthetic pathway leading to fura-1, fura-2, and fura-3.
- FIG. 3 is a graph showing a family of excitation spectra for fura-2 at 20° in buffers with free Ca 2+ values ranging from ⁇ 1 nM to >10 microM.
- FIG. 4 is a graph showing emission spectra for indo-1 as a function of free Ca 2+ .
- FIG. 5 is a graph showing excitation spectra for 1 microM fura-2 and 30 microM quin2 under identical sets of conditions.
- FIG. 6 is a graph showing the effect of Mg 2+ on fura-2.
- FIG. 7 is a graph showing the effect of pH excursion between 6.75 and 7.05 on fura-2 at zero, 200 nM and 1 mM free Ca 2+ .
- FIG. 1 shows the synthetic pathway leading to stil-1, stil-2, and indo-1.
- the Roman numerals used in the figure are keyed to the synthetic details discussed in the METHODS OF SYNTHESIS section, infra.
- FIG. 2 shows the synthetic pathway leading to fura-1, fura-2, and fura-3. Again, the Roman numerals used in the figure are keyed to the synthetic details discussed in the METHODS OF SYNTHESIS section, infra.
- FIG. 3 shows a family of excitation spectra for 1 microM fura-2 at 20° C. in buffers with free Ca 2+ values ranging from ⁇ 1 nM to >10 microM.
- the excitation bandwidth was 1.9 nm
- the emission was collected at 510 nm with 4.6 nm bandwidth
- the spectra were automatically corrected for excitation lamp and monochromator characteristics using a rhodamine B quantum counter.
- the titration was done starting with 5 ml of 100 mM KCl, 10 mM KMOPS, 10 mM K 2 H 2 EGTA, 1 microM fura-2, adjusting the pH to 7.20, recording the spectrum, then discarding 0.5 ml of this solution and replacing with 0.5 ml of 100 mM KCl, 10 mM KMOPS, 10 mM K 2 CaEGTA, 1 microM fura-2, readjusting the pH to 7.20, and recording the spectrum which was then in 9 mM K 2 H 2 EGTA and 1 mM K 2 CaEGTA.
- FIG. 4 shows emission spectra for indo-1 as a function of free Ca 2+ .
- the dye, 6 microM was titrated in 115 mM KCl, 20 mM NaCl, 10 mM MOPS, 1.115 mM MgCl 2 , 1.115 mM K 2 H 2 EGTA, KOH to pH 7.050 ⁇ 0.004, 37° C., to which small aliquots of K 2 CaEGTA were added from a micrometer syringe to raise free Ca 2+ to the values labeling each curve, in units of nM unless otherwise specified.
- Excitation was at 355 nm, and both excitation and emission were set to 5 nm bandwidth.
- FIG. 5 shows excitation spectra of 1 microM fura-2 and 30 microM quin-2 under identical sets of conditions: 130 mM KCl, 10 mM MOPS, 1 mM EDTA, KOH to pH 7.20, 20° C., before and after addition of 2 mM CaCl 2 to give 1 mM free Ca 2+ . Emission was collected at 500 nm, 4.6 nm bandwidth, and the excitation bandwidth was 0.93 nm. Quin2 free acid was from Lancaster Synthesis, Morecambe, England.
- FIG. 6 shows the effect of Mg 2+ on fura-2.
- An excitation spectrum was recorded of 2 microM fura-2 in 2.5 ml 32 mM KCl, 1 mM EGTA, 10 mM MOPS, adjusted with KOH to pH 7.18 while thermostatted at 20° C. Then 25 microliters of this solution was discarded and replaced by 25 microliters of a solution containing 2 microM fura-2, 104.8 mM MgCl 2 , 5.8 mM EGTA, 10 mM MOPS, adjusted with KOH to pH 7.18. Since both stocks had 1 mM free EGTA but the latter had 100 mM free Mg, the 99:1 v/v composite went to 1.0 mM free Mg 2+ .
- FIG. 7 shows the effect of pH excursion between 6.75 and 7.05 on fura-2 at zero, 200 nM, and 1 mM free Ca 2+ .
- An excitation spectrum of 0.5 microM fura-2 was recorded in 135 mM KCl, 5 mM MOPS, 2 mM EGTA, adjusted with KOH to pH 7.05 at 37°.
- the succeeding spectra were taken following acidification with HCl to pH 6.75; addition of 0.379 mM CaCl 2 and KOH to pH 6.75; addition of 0.655 mM more CaCl 2 and KOH to pH 7.05; addition of 2 mM CaCl 2 and KOH to pH 6.75; KOH to pH 7.05.
- [Ca 2+ ] i means intracellular free calcium.
- EGTA means ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetracetic acid.
- BAPTA means 1,2-bis(2-aminophenoxy)ethane N,N,N',N'-tetraacetic acid; the chemical structure for BAPTA is: ##STR1##
- BAPTA-like means substituted derivatives of BAPTA which retain the essential characteristic of two bis(carboxymethyl)amino-substituted phenyl rings, said rings being linked at the positions ortho to the amines through a four atom bridge wherein the atom adjacent to each phenyl ring is N or O and the two center atoms are each C.
- BAPTA-like includes compounds like quin-1 and quin-2.
- quinl means 2-[[2-[bis(carboxymethyl)amino]-5-methylphenoxy]methyl]-8-[bis(carboxymethyl)amino]-quinoline.
- quin2 means 2-[[2-[bis(carboxymethyl)amino]-5-methylphenoxy]-6-methoxy-8-[bis(carboxymethyl)amino]quinoline; the chemical structure for quin2 is: ##STR2##
- MOPS 3-(N-morpholino)propanesulfonic acid.
- stil-1 means 1-(2-amino-5-[(2-(4-carboxyphenyl)-E-ethenyl-1-]phenoxy)-2-(2'-amino-5'-methylphenoxy)ethane-N,N,N',N'-tetraacetic acid.
- the chemical structure for stil-1 is shown in FIG. 1 as compound X.
- stil-2 means 1-(2-amino-5-[(2-(4-N,N-dimethylaminosulfonylphenyl)-E-ethenyl-1-]phenoxy)2-(2'-amino-5'-methylphenoxy)ethane-N,N,N', N'-tetraacetic acid.
- the chemical structure for stil-2 is shown in FIG. 1 as compound XII.
- indo-1 means 1-(2-amino-5-[6-carboxyindolyl-2]-phenoxy)-2-(2'-amino-5'-methylphenoxy)ethane-N,N,N',N'-tetraacetic acid.
- the chemical structure for indo-1 is shown in FIG. 1 as compound XVII.
- fura-1 means 1-(2-(4-carboxyphenyl)-6-amino-benzofuran-5-oxy)-2-(2'-amino-5'-methylphenoxy)ethane-N,N,N',N'-tetraacetic acid.
- the chemical structure for fura-1 is shown in FIG. 2 as compound XXIX.
- fura-2 means 1-(2-(5'-carboxyoxazol-2'-yl)-6-aminobenzofuran-5-oxy)-2-(2'-amino-5'-methylphenoxy)ethane-N,N,N',N'-tetraacetic acid.
- the chemical structure for fura-2 is shown in FIG. 2 as compound XXXV.
- fura-3 means 1-(2-(4-cyanophenyl)-6-aminobenzofuran-5-oxy)-2-(2'-amino-5'-methylphenoxy)ethane-N,N,N',N'-tetraacetic acid.
- the chemical structure for fura-3 is shown in FIG. 2 as compound XXXII.
- esters mean those readily hydrolyzable esters which are known and used in the pharmaceutical industry, especially alpha-acyloxyalkyl esters. See generally, references 37 and 38.
- microM means micromolar
- FIGS. 1 and 2 The chemical formulas for compounds shown in FIGS. 1 and 2 are identified with Roman numerals. These Roman numerals are used throughout the specification (see especially, the METHODS OF SYNTHESIS section, infra) to identify compounds that correspond to those shown in the figures.
- the new dyes disclosed herein are named with hyphens to distinguish the number 1 from the letter l, e.g., indo-1, fura-1, fura-2, stil-1, stil-2, etc.
- the present invention comprises a new class of fluorescent, calcium-specific indicator dyes that combines a stilbene-type fluorophore with a tetracarboxylate Ca 2+ chelating compound having the octacoordinate ligating groups characteristic of EGTA and BAPTA.
- the new dyes are comprised of a BAPTA-like Ca 2+ chelator coupled to a single fluorescent dye.
- the new dyes are comprised of a bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid having the generic formula: ##STR3## and the pharmaceutically acceptable non-toxic salts and esters thereof wherein:
- R 1 is selected from the group comprised of --H, --CH 3 , --F, --Cl, and --Br;
- R 2 is selected from the group comprised of --H, --CH 3 , --F, --Cl, --Br, and C 0 -C 4 alkoxy;
- R 3 or R 3a are independently H or a polar, electron withdrawing function selected from the group comprised of --CO 2 H, --CO 2 R 4 , --CONR 5 R 6 , --SO 3 H, --SO 2 NR 5 R 6 , --SO 2 CF 3 , --COCH 3 , and --CN, or
- R 3 and R 3a together are --(CO)NR 5 (CO)--;
- R 4 is alpha-acyloxyalkyl or a pharmaceutically acceptable esterifying group
- R 5 and R 6 are independently --H, methyl, or ethyl
- Z is an aromatic or heteroaromatic system coupled to the 5 position of one 2-aminophenoxy ring through a trans ethylenic linkage, wherein said ethylenic linkage may itself be part of an aromatic heteroaromatic ring system;
- W is H and X is H;
- W and X together are 0, or NR 5 ;
- Y is H; Y' is H (unless A is NR 5 , O, or S); or
- Y and Y' together are NR 5 and A is C;
- a and A' are independently C, N, O, or S;
- D is O, N, NH, or N alkyl
- Q is a double bond when D is N and a single bond otherwise;
- R 7 is H and R 8 is H or
- the new dyes are comprised of a BAPTA-like Ca 2+ chelator coupled to a pair of fluorescent dyes.
- the new dyes are comprised of a compound having the generic formula: ##STR4## and the pharmaceutically acceptable non-toxic salts and esters thereof wherein:
- R 3 or R 3a are independently H or a polar, electron withdrawing function selected from the group comprised of --CO 2 H, --CO 2 R 4 , --CONR 5 R 6 , --SO 3 H, --SO 2 NR 5 R 6 , --SO 2 CF 3 , --COCH 3 , and --CN, or
- R 3 and R 3a together are --(CO)NR 5 (CO)--;
- R 4 is alpha-acyloxyalkyl or a pharmaceutically acceptable esterifying group
- R 5 and R 6 are independently --H, methyl, or ethyl
- Z is an aromatic or heteroaromatic system coupled to the 5 position of one 2-aminophenoxy ring through a trans ethylenic linkage, wherein said ethylenic linkage may itself be part of an aromatic heteroaromatic ring system;
- W is H and X is H;
- W and X together are O, or NR 5 ;
- Y is H; Y' is H (unless A is NR 5 , O, or S); or
- Y and Y' together are NR 5 and A is C;
- a and A' are independently C, N, O, or S;
- D is O, N, NH, or N alkyl
- Q is a double bond when D is N and a single bond otherwise;
- R 7 is H and R 8 is H or
- Preferred indicators in both the first and second forms contain heterocyclic bridges to restrict the ethylenic bond of the stilbene to the trans configuration and to reduce hydrophobicity.
- the present invention comprises an improved method for synthesizing alpha-acyloxyalkyl bromides, especially acetoxymethyl bromide which is useful in preparing the acetoxymethyl esters contained within the class of new indicators disclosed and claimed herein.
- Alpha-acyloxyalkyl bromides are useful to the pharmaceutical industry in the preparation of prodrugs (see references 38 and 39).
- the improved method comprises reacting approximately 1 equivalent of 1,1-diacyloxyalkane and approximately 1 to 1.5 equivalents of trimethylsilyl bromide, in the presence of approximately 0.1 to 0.2 equivalents of a Lewis acid catalyst, in a reaction vessel with stirring until the reaction is complete, and then recovering the alpha-acyloxyalkyl bromides so produced.
- Alpha-acyloxyalkyl bromides produced by known methods are contaminated with bis(1-bromoalkyl) ether.
- Alpha-acyloxyalkyl bromides produced by the improved method of the present invention are free of contaminating bis(1-bromoalkyl) ether.
- the new method is capable of making certain alpha-acyloxyalkyl bromides that would be extremely difficult or impossible to make by known methods because those known methods require pure anhydrous aldehydes that are hygroscopic, unstable, and prone to polymerization.
- the present invention comprises a new class of fluorescent, calcium-specific indicator dyes that combines a stilbene-type fluorophore with analogs of the octacoordinate tetracarboxylate Ca 2+ chelating compound, BAPTA.
- the new dyes are comprised of a BAPTA analog coupled to a single fluorescent dye; in a second form, a BAPTA analog is coupled to a pair of fluorescent dyes.
- Preferred compounds in both the first and second forms contain heterocyclic bridges to restrict the ethylenic bond of the stilbene to the trans configuration.
- the new indicator dyes disclosed herein have in common at least one stilbene-type functionality attached to at least one of the aromatic rings in the parental BAPTA-like compound.
- This stilbene-type of function consists of an aromatic or heteroaromatic system that is coupled to a parental (BAPTA-like) aromatic ring through two carbons containing a trans double bond.
- the fluorophore is electronically coupled to the chelator, when calcium is bound by the molecule, the chelating portion of the molecule no longer donates electron density to the fluorophore portion, causing a decrease in maximum wavelength of absorbance. As long as there is no more than one sigma bond in a row in the link between fluorophore and BAPTA-like chelator, this electrical coupling will be maintained.
- the link between the fluorophore and chelator portions can also be part of a complex heterocycle, which in turn can be part of the fluorophore or part of the chelator.
- cation affinities of BAPTA derivatives can be increased or decreased by appropriate electron-donating or electron-withdrawing substituents (see generally reference 6).
- substitution of two methyl groups for the two hydrogens para to the two nitrogens of BAPTA strengthened Ca 2+ binding by 0.4 log unit and similar disubstitution with bromine weakened Ca 2+ binding by 1.2 log units (see reference 6).
- monosubstitution gives half as much effect on calcium affinity (data not shown).
- R 2 is anything except hydrogen
- the 5 position of that ring will be blocked and formylation can proceed only at the desired 5 position of the 4-W-benzene ring or equivalent quinoline.
- R 2 is hydrogen
- the presence of halogen at R 1 will deactivate that ring for electrophilic substitution and formylation will occur mainly at the desired position.
- the desired product can be prepared by controlled formylation and separation of the desired product or by beginning the synthesis with an appropriate 3-hydroxy-4-nitrobenz- aldehyde (see METHODS OF SYNTHESIS section, infra).
- R 2 must be hydrogen and R 1 can be hydrogen, alkyl or any variation of substituent W.
- R 1 can be hydrogen, alkyl or any variation of substituent W.
- the desired formylation occurs at the 5 position of both phenyl rings or the 5 position of both the R 1 , R 2 phenyl and of the quinoline or benzopyran.
- the dye component of the molecules is based on trans stilbene, but can take many other forms.
- the ethylene bridge of stilbene can be locked in the trans configuration by forming a heterocyclic ring which includes the bridge.
- Y and Y' in the generic formulas shown in the BRIEF DESCRIPTION OF THE INVENTION section, supra, can together be a heteroatom, such as NR 5 and the dye molecule can be indole.
- the synthesis of such an indole is described infra in the METHODS OF SYNTHESIS section to illustrate this class of dye components (see indo-1).
- a heterocycle can also be formed which locks the ethylenic bridge to the phenyl ring of the chelating portion of the molecule.
- connection can be trans-ethylene or part of a heterocycle, as discussed above.
- Z phenyl or oxazole
- Z compounds which demonstrate use of phenyl or oxazole as Z, e.g. compounds stil-1 or fura-2, respectively, or, in conjunction with Y and Y 1 and the ethylenic bridge, indole as Z, e.g. compound indo-1.
- R 3 and R 3a substituents of Z serve to increase the hydrophilicity of the aromatic, generally hydrophobic, dye component of the final molecule.
- R 3 and R 3a also can be used to adjust somewhat the maximum absorbance or emission wavelength of the dye.
- a wide variety of R 3 and R 3a substituents can be incorporated in the dyes disclosed and claimed in this invention, including the cyclic imide formed when R 3 and R 3a together are --(CO)NR 5 (CO)--.
- carboxy, cyano, and aminosulfonyl for R 3 are illustrated (see synthesis of compounds fura-1, fura-3, and stil-2, respectively).
- Example I discusses absorbance properties for the new indicators in the presence and absence of Ca 2+ while Example II discusses the fluorescence properties.
- Example III discloses Ca 2+ binding constants. Photoisomerization of the stilbene-type dyes is discussed in Example IV while cation selectivity is discussed in Example V.
- Example VI discusses membrane binding and Example VII discloses calibration equation for a dye using fluorescence intensity values at two wavelengths.
- increased brightness of fluorescence is the most dramatically noticeable advantage of the new dyes as compared with their immediate predecessors such as quin2.
- the new dyes are ten to thirty-fold more strongly fluorescent, partly because of their five-to-six-fold higher extinction coefficients and partly because of their higher quantum efficiencies of fluorescence (up to 0.5 as compared with about 0.1 for quin2).
- the increased fluorescence means that far less dye needs to be introduced into a given amount of cells to get an adequate signal. The less dye inside the cells, the less likelihood there is of significantly buffering the calcium levels being measured or otherwise perturbing the cells.
- the increased brightness can permit Ca 2+ measurement in shorter observation times or from smaller volumes of tissue.
- the next most important feature of the new dyes is the ability of Ca 2+ to significantly change the shape and wavelength distribution of the fluorescence spectra, not just the intensity. This wavelength shift makes it possible to measure calcium concentrations on an absolute scale simply from the ratio of fluorescences at two wavelengths, without consideration of dye concentration, optical pathlength, or absolute sensitivity of detection. (See Example VII.) All six of the preferred dyes (stil-1, stil-2, indo-1, fura-1, fura-2 and fura-3) disclosed herein as examples of the present invention have this feature in their excitation spectra.
- indo-1 is so far unique in also having a major change in emission wavelength upon binding calcium.
- Existing dyes such as quin2 change intensity but not peak wavelength upon binding calcium, making their signals relatively difficult to calibrate. As a result they are not as useful as the new dyes disclosed herein for measuring calcium concentrations on an absolute scale by means of the wavelength ratio method.
- both the wavelengths and Ca 2+ dissociation constant should increase when groups more strongly electron-withdrawing than carboxylate are placed at the far end of the chromophore (compare fura-1 and fura-3).
- the terminal carboxylate is at least theoretically attractive as a deterrent of hydrophobic bonding of the long chromophore tail.
- a fifth advantage of the new dyes over known dyes such as quin2 is their better selectivity for Ca 2+ over divalent cations such as Mg 2+ , Mn 2+ , Zn 2+ and Fe 2+ .
- the improvements for fura-2 over quin2 are about three-fold, twelve-fold, and forty-fold for Mg 2+ , Mn 2+ , and Zn 2+ respectively.
- Most workers using quin2 have implicitly assumed that cytosolic free Mg 2+ was 1 mM as was measured in lymphocytes (see reference 7).
- the true free [Mg 2+ ] were 0 or 2 mM respectively, the true ⁇ Ca 2+ ] values would be about 0.5 or 1.5 times higher than those estimated assuming 1 mM [Mg 2+ ].
- the new dyes should be less sensitive yet to uncertainties in [Mg 2+ ].
- the only clear instance of heavy metal interference with quin2 has been in certain tumor cell lines (see reference 8).
- the reduced heavy metal affinities of the new dyes should help guard against other occurrences as well as toxicity due to chelation of essential heavy metals.
- These dyes and quin2 might even be used as heavy metal indicators by working at the wavelengths where all the different Ca 2+ curves cross each other.
- the improved divalent cation selectivity of the new dyes is probably due to the abandonment of the quinoline structure with its troublemaking ring nitrogen (see reference 6).
- Magnesium binding affects the spectrum much less than Ca 2+ -binding probably because Mg 2+ is too small to bind to both amino nitrogens at once (see reference 6) and tends to choose the nitrogen on the right side of the structure as drawn, since that one is not in conjugation with the slightly electron-withdrawing chromophoric skeleton.
- Fura-2 and EGTA both respond to increasing temperature by weakening their Ca 2+ affinity and increasing their Mg 2+ affinity (see reference 11). This suggests that the thermodynamic bases for their Ca 2+ :Mg 2+ selectivities are similar.
- fura-2 will be the preferred dye for most applications although indo-1 is the prime candidate for flow cytometry.
- the stilbenes are marginally easier to synthesize but do not fluoresce as brightly as the others.
- the stilbenes have complicated problems of photoisomerization and photodecomposition and are relatively difficult to load as acetoxymethyl esters (data not shown) due to insufficient solubility. It remains to be seen whether the slightly longer wavelengths of fura-3 can compensate for its lower brightness and less hydrophilic chromophore.
- the new dyes have already made [Ca 2+ ] i measurements possible in a number of previously intractable systems. The prospect of studying individual cells or aggregated tissue should enormously expand the range and ease of optical measurement of [Ca 2+ ] i and its ubiquitous messenger functions.
- Methods for synthesizing the new dyes disclosed herein include the following:
- the present invention comprises a new class of fluorescent calcium-specific indicator dyes.
- Preferred examples of these new dyes include stil-1, stil-2, fura-1, fura-2, fura-3 and indo-1.
- FIGS. 1 and 2 outline the synthetic routes utilized in preparing these compounds. Full descriptions of the reaction conditions are described below. In both the figures and the discussion that follows, Roman numerals are used to identify the various chemical compounds.
- a methyl, halogen, or alkoxy group as radical R 2 prevents electrophilic substitution at that position, restricting formylation to the R 1 -R 2 -phenyl ring. Even if R 2 is hydrogen, a halogen at position R 1 will deactivate for electrophilic substitution the right hand benzene ring of the compound 1a, so that little or no formylation will occur on that ring.
- a methyl group at R 1 may slightly promote formylation at the R 2 position, but any doubly formylated impurities should be readily removable.
- R 2 is H and W is an O or NR 5 precursor, the ability of those W groups to promote electrophilic substitution is sufficient to direct formylation preferentially to the 5-position ortho to W. Since 8-aminoquinoline systems formylate much more readily than phenyl systems, no claimed combination of R 1 and R 2 will prevent the straightforward formylation of compounds containing quinoline or benzopyran systems.
- the same 5-formyl compound could be prepared by using an acetal of 3-hydroxy-4-nitro benzaldehyde in place of compound III and 2-nitro-4-R 1 -5-R 2 -phenoxide in place of compound I and following the steps illustrated below for the preparation of compound VI (condensation with 1,2-dibromo-ethane, reduction, and alkylation with alkylbromoacetate). Mild acid hydrolysis of such an analogue of compound VI would unmask the aldehyde to give monoaldehyde, 1a.
- Aldehydes 1b which are derivatives of salicylaldehyde when W is OH, are synthesized in eight steps from p-hydroquinone. Key steps in the preparation of the salicylaldehyde include the acid-catalyzed, selective debenzylation (see reference 20) of the phenolic group ortho to the nitro in preference to the phenolic group meta to nitro. The resulting 2-nitro-4 benzyloxyphenol can then be used as a starting material in the procedure outlined above to prepare the key aldehyde, 1b. Attempts to convert XXIV to XXV by hydroxide displacement of benzyloxide were unsuccessful.
- a phenol can be condensed with 1-bromo-2-(2-nitro-4-R 1 -5-R 2 -phenoxy)ethane, and reduced (H 2 /Pt). Any of nitro, nitroso or diazonium salt groups will thereby be reduced to NH 2 without affecting the benzyloxycarbonyl groups.
- the methoxy group can be cleaved with BBr 3 or hot HI to yield a 6-hydroxy group, which can then be protected by reaction with dimethoxymethane under acid catalysis to yield 2-methyl-6-methoxymethyloxy-8-nitroquinoline.
- dialdehyde shown below as compound 1d or its analogs.
- the dialdehyde By using a single or two different 2-nitrophenols with no 5 substituent and hydrogen, alkyl, or benzyloxy as a 4 substituent and following the synthesis outlined for aldehyde 1b, the Vilsmeier formylation step will produce the dialdehyde, 1d. This dialdehyde can then be condensed with any of the Wittig reagents used with 1a, leading to final compounds containing two long-wave fluorophores.
- compounds can be prepared which contain a heterocyclic ring to lock the ethylene bridge in the trans configuration by connecting the ethylene to the rest of the dye portion of the molecule.
- the synthesis of indo-1, XVII, is described, infra.
- the aldehyde of choice is condensed with a Wittig reagent carrying an ortho nitro group, which can then be reduced with triethyl phosphite to generate a nitrene, which cyclizes immediately to an indole (see reference 19).
- Compounds can also be prepared which lock the ethylene bridge in the trans configuration by connecting the ethylene bridge to the chelating portion of the molecule.
- the aldehyde is condensed with an appropriate halomethyl aromatic compound, for example, compounds such as XXVII, XXX or XXXIII (see FIG. 2).
- Ring closure with a base such as potassium fluoride on alumina leads to the desired indole, by analogy to XXVII, XXXI and XXXIV.
- R 3 and R 3a are present when needed to provide hydrophilicity to the otherwise hydrophobic fluorophore "tail". R 3 and R 3a will also modify somewhat the fluorescent properties of the long wave chromophore.
- Z groups include heterocycles containing quaternary nitrogen, such as pyridinium.
- Z groups of this type do not require an R 3 or R 3a substituent to reduce hydrophobicity of the fluorphore, although R 3 or R 3a may still be useful to modify the fluorescence wavelength of the fluorophore.
- trifluoroacetoxymethyl bromide CF 3 COOCH 2 Br
- CF 3 COOCH 2 OCOCF 3 trifluoroacetoxymethyl bromide
- benzoyloxymethyl bromide C 6 H 5 COOCH 2 Br
- was prepared from methylene dibenzoate C 6 H 5 COOCH 2 OCOC 6 H 5
- 1-acetoxy-1-bromoacetone was prepared from 1,1-diacetoxyacetone, itself prepared by action of sodium acetate on 1,1-dichloracetone.
- 1-acetoxy-1-bromoacetone was extremely difficult or impossible to make by the standard preparation of 1-acyloxyalkyl bromide compounds, since the standard route would require preparation of pure anhydrous pyruvaldehyde, CH 3 COCHO, to be reacted with acetyl bromide.
- Anhydrous pyruvaldehyde is extremely hygroscopic, unstable, and prone to polymerization.
- 1-acyloxyalkyl chlorides can also be obtained by this general procedure, though more vigorous conditions are required and lower yields result.
- C 6 H 5 COOCH 2 OCOC 6 H 5 and Me 3 SiCl reacted in CCl 4 with AlCl 3 catalysis at 100° (sealed vessel) for 60 hrs. and gave a 35% yield of C 6 H 5 COOCH 2 Cl.
- Alpha-acyloxyalkyl bromides prepared by the above method are useful in preparing esters of the flourescent dyes disclosed and claimed herein.
- acetoxymethyl esters of the compounds claimed herein were best made by: saponifying the methyl or ethyl esters of the target chelator (for example, compounds IX, XII, XVI, XXVIII, XXXI, or XXXIV, below and in FIGS. 1 and 2) with KOH, adding four to six equivalents of a tetrabutylammonium salt (preferably the hydroxide), and evaporating in vacuo to dryness.
- the target chelator for example, compounds IX, XII, XVI, XXVIII, XXXI, or XXXIV, below and in FIGS. 1 and 2
- Potassium salts of 2-nitrophenoxides were prepared by adding a stoichiometric amount of concentrated aqueous KOH to the nitrophenol in methanol, then evaporating the solvent in vacuo until the stoichiometric weight was reached.
- the phosphonium salt (VIII) was prepared in 92% yield by reacting 12.16 g (50 mM) ethyl p-bromomethylbenzoate, XXVII (see reference 30), with 14.48 g (55 mM) triphenylphosphine in refluxing toluene overnight. The precipitate was filtered off from the cooled reaction mixture. After washing, and drying it had m.p. 241°-243° and weighed 23.3 g, 92% yield and was sufficiently pure for further use.
- Sulfonamide phosphonium salt XI was prepared analogously to VIII by reacting triphenylphosphine with N,N-dimethyl 4-(chloromethyl)benzenesulfonamide in refluxing toluene. Its m.p. was 275°-280°.
- the sulfonamide was prepared by reacting 4-chloromethyl benzenesulfonyl chloride (see reference 31) with a cold solution of dimethylamine in ether.
- Phosphonium salt XIV (m.p. 165-168) was prepared in 76% yield by reacting 1.5 g triphenylphosphine with 1.2 g ethyl 4-bromomethyl-3-nitrobenzoate in refluxing toluene overnight.
- the latter ester was prepared by stirring 2.43 g of ethyl p-bromomethylbenzoate, XXVII, in 10 ml concentrated H 2 SO 4 at 5° C., while adding 1 ml conc HNO 3 (d 1.4) dropwise.
- the nitration mixture was poured onto crushed ice and extracted with CH 2 Cl 2 . The extract was washed with dilute NaOH, dried, and evaporated to give an oil which eventually crystallized, m.p.
- the cooled reaction mixture was evaporated to dryness, diluted with toluene and filtered; the gray precipitate was washed repeatedly with further small portions of toluene.
- the pooled toluene solutions were repeatedly extracted with phosphate buffer until the washings were pH 2, the same as the fresh buffer.
- the toluene solution was then washed with water and dried over MgSO 4 .
- After evaporation of the toluene under reduced pressure, the residue was washed with petroleum ether-ethyl acetate 9:1 v/v to remove traces of ethyl bromoacetate.
- the yield of XXIV was 6.4 g (90%), m.p. 95°-96°.
- XXV was debenzylated by hydrogenation in glacial acetic acid with 5% Pd on charcoal catalyst at atmospheric pressure.
- XXVI had m.p. 109°-111°.
- ether formation and furan ring closure can be done in one step.
- 0.5 (g) (0.77 mmole) XXVI, 0.2 g ethyl 2-chloromethyloxazole-5-carboxylate (XXX), and 0.5 g K 2 CO 3 were stirred in 2 ml dry dimethylformamide at 100° for 1.5 hr.
- the reaction mixture was cooled, diluted with water, acidified with HCl and extracted with CHCl 3 .
- the combined extracts were dried with MgSO 4 and evaporated to a dark, semicrystalline residue.
- Ethyl 2-chloromethyloxazole-5-carboxylate (XXX) was prepared by reacting chloracetonitrile with ethyl diazopyruvate in analogy to the synthesis (see reference 34) of ethyl 2-methyloxazole-5-carboxylate.
- Ethyl diazopyruvate, 0.1 g, a few mg of cupric acetylacetonate, and 0.4 ml of chloracetonitrile were kept overnight at 60° in benzene solution. The cooled reaction mixture was partitioned between aqueous NaHCO 3 and ether.
- the solid free acids are not stable for more than a few days, though their fully ionized salts are considerably more stable.
- the final products were not characterized by NMR, but were carefully studied by UV, fluorescence, and Ca 2+ titration (see Table 1). The Ca 2+ titration showed that no preparation contained more than 10% di- or tricarboxylate compounds (data not shown).
- XXXII 1-(2-(5'-carboxyoxazol-2'-yl)-6-amino-benzofuranoxy-5-)-2-(2'-amino-5'-methylphenoxy)ethane-N,N,N',N'-tetraacetic acid
- XXXV 1-(2-(4-cyanophenyl)-6-amino-benzofuranoxy-5-)- 2-(2'-amino-5'-methylphenoxy)ethane-N,N,N',N'tetraacetic acid
- esters are saponified to free chelator polyanions by dissolving the ester in a water-miscible solvent such as methanol, ethanol, or dioxane and adding excess aqueous sodium, potassium, or tetrabutylammonium hydroxide.
- a water-miscible solvent such as methanol, ethanol, or dioxane
- Acetoxymethyl bromide, CH 3 COOCH 2 Br was prepared by stirring 4.0 g (30 mM) methylene diacetate and 5.0 g (33 mM) trimethylsilyl bromide, in the presence of 60 mg anhydrous ZnCl 2 but no added solvent, for 48 hr at room temperature.
- the product was distilled crudely at 10 torr, 40°-60° then refractionated through a Vigreaux column at 10 torr, 55°-57°, yielding 4.04 9, 87%, containing no detectable BrCH 2 OCH 2 Br by NMR.
- Lewis acid catalysis is essential; without it there is no reaction even at 110° in a sealed tube overnight.
- Methylene diacetate was prepared from paraformaldehyde and acetic anhydride (see reference 35) and was carefully distilled to remove CH 3 COOCH 2 OCH 2 OCOCH 3 .
- UV absorbance spectra were recorded on a Cary 210 spectrophotometer at 22° ⁇ 2° C. Fluorescence spectra were recorded either on a Perkin-Elmer MPF-44A spectrofluorometer or on a Spex Fluorolog 111, both in ratio mode with Rhodamine B quantum counters. Correction factors for the emission monochromator and photomultiplier were obtained by comparing the spectra of reference compounds with their known quantum distributions (see reference 9). The quantum yields were determined by comparing the integral of the corrected emission spectrum of the test sample with that of a solution of quinine bisulfate in 1N sulfuric acid. The concentration of quinine was adjusted to give the same absorbance as the sample at the exciting wavelength. The quantum efficiency of quinine was taken to be 0.55 (see reference 10).
- EGTA calcium and magnesium affinities of EGTA at high ionic strength were measured by titrating 125 micromole portions of EGTA (Fluka Chemical, puriss grade) with 1.00M KOH (J.T. Baker, low in carbonate).
- the EGTA was dissolved in 25 ml medium containing 225 mM KCl, 25 mM NaCl, and in some runs 10 mM CaCl 2 or 21 mM MgCl 2 . This solution was stirred in a Radiometer TTA80 Titrator, bubbled continuously with nitrogen, and thermostatted at 18° C. while KOH was added in 0.005 or 0.01 ml aliquots from a ABU80autoburet.
- the pH was measured with a PHM84 meter, a G2040C electrode and an Ag/AgCl reference electrode connected to the titration solution by a long length of 0.01 inch catheter tubing filled with 3M KCl; this electrode chain was calibrated with phthalate (4.001), phosphate (6.889), and borax (9.245) buffers made according to National Bureau of Standards recipes.
- the fifty to eighty pH readings in each titration were analyzed by the FORTRAN program SCOGS of Sayce (see reference 36).
- the resulting proton ionization constants for EGTA were 9.537 and 8.961 with computed uncertainties of ⁇ 0.012 and ⁇ 0.008 respectively. These pK a values are "mixed" constants, i.e.
- K' Ca the effective stability constant K' Ca may be calculated as (antilog 10.77) ⁇ [1 +antilog (3.51-pH)]/[1+antilog (9.537-pH)+antilog (9.537+8.961-2 ⁇ pH)]in units of liters/mole.
- K' Mg (antilog 4.567) ⁇ [1+antilog (7.885-pH)]/[1+antilog (9.537-pH)+antilog (9.537+8.961-2 ⁇ pH)].
- Free [Ca 2+ ] levels were controlled by Ca-EGTA buffers assuming apparent dissociation constants for Ca-EGTA of 380 nM at pH 7.00 in 0.1M KCl, 20° C.; 151 nM at pH 7.20 in 0.1M KCl, 20° C.; 214 nM at pH 7.05 in 130 mM KCl, 20 mM NaCl at 37° C.; and 490 nM at pH 7.02 in 225 mM KCl, 25 mM NaCl at 18° C.
- the first three apparent dissociation constants were calculated from the tables of Martell & Smith (see reference 11) as discussed in referencess 1 and 12.
- Free [Mg 2+ ] was likewise controlled by Mg-EGTA buffers assuming an apparent dissociation constant for Mg-EGTA of 21 mM at pH 7.18 in 130 mM KCl at 20°, 8.96 mM at pH 7.05 in 130 mM KCl, 20 mM NaCl at 37°, and 99 mM at pH 7.02 in 225 mM KCl, 25 mM NaCl at 18°. Again, the last value was freshly determined in this study.
- Heavy metal binding constants were measured as follows. Affinities of stil-1 and fura-2 for Zn 2+ were measured by titrating 1-2 microM dye in 120 mM KCl, 5 mM K 2 H 2 EGTA, 10 mM MOPS, 5 mM Tris, with 1M ZnCl 2 dispensed from a micrometer syringe. At each stage of the titration (13 steps from 0 to 7 mM total ZnCl 2 added) the pH was adjusted with base to be within 0.02 units of 7.12, the actual pH values was noted for computation of pZn, and the excitation spectrum was recorded at room temperature. The family of spectra had a roughly similar appearance to FIG.
- One series also contained 0, 0.1, or 1 mM MnSO 4 and 0, 1.1, 2.2, 4.4, or 8.8 mM CaCl 2 ; another series had 2 mM CaCl 2 and 0, 0.1, 0.2, 0.5, 1, 2 mM Fe(NH 4 ) 2 (SO 4 )2; and a third series with 0.2 mM Fe(NH 4 ) 2 (SO 4 ) 2 and 0, 0.2, 0.4, 0.8, 2, 4, and 8 mM CaCl 2 .
- Binding to membranes was assessed by adding 0.3 ml portions of well-washed, packed human red cells to 1.5 ml volumes of 2 microM fura-2, fura-3, or indo-1, in media containing 145 mM NaCl, 5 mM KCl, 10 mM MOPS, 1 mM Na 2 HPO 4 , 1 mM MgCl 2 , and either 1 mM EGTA or 1 mM CaCl 2 . After several minutes equilibration, the suspensions were centrifuged and the fluorescence of the supernatant was measured with front-face geometry.
- the fluorescence excitation spectra for the new indicators disclosed herein shift to shorter wavelengths as [Ca 2+ ] increases, much as the absorption spectra do.
- FIG. 3 presents a set of excitation spectra for fura-2 in calibration buffers of varied [Ca 2+ ].
- the [Ca 2+ ] in an unknown solution containing the dye can be deduced from the shape of the excitation spectra.
- An explicit formula for this calibration is derived in Example VII.
- indole-type emission spectra show a clear crossover point whose position depends on the excitation wavelength because that affects the relative efficiency with which Ca 2+ -free and Ca 2+ -bound forms are excited. This is illustrated in FIG. 4. That figure also shows that an excitation wavelength of 355-356 nm (as would be produced by a krypton laser), indo-1 gives the family of emission spectra in which the long and short wavelength peaks are of comparable amplitude.
- the quantum yields of fluorescence for all the new indicators are quite respectable, ranging up to ca. 0.5 for indo-1 and fura-2 with Ca 2+ .
- the Ca 2+ -complex has a quantum efficiency between 1.3 and 2.1-fold higher than the Ca 2+ -free dye.
- the quantum efficiencies of quin2 with and without Ca 2+ are 0.14 and 0.029 respectively (see reference 6). Since fura-2 at half-saturation has a quantum efficiency four to five times higher than quin2 at half-saturation, and fura-2 has extinction coefficients about six times higher than quin2, one would predict that fura-2 should be some thirty-fold brighter than quin2. This estimate is confirmed by FIG. 5, which shows that the fluorescence of 1 microM fura-2 at high and low Ca 2+ is bracketed by that of 30 microM quin2 under identical conditions.
- Dissociation constants for the new indicators disclosed herein are in the range 100-300 nM at ionic strengths near 0.1 to 0.15. Such values are close to the dissociation constant (110 nM) of the parent compound BAPTA (see reference 6), showing that the substituents that make up the rest of the chromophores are only slightly electron-withdrawing. Fura-2 was also examined at higher ionic strengths, ca. 250 mM (225 mM KCl, 25 mM NaCl). A considerable decrease in Ca 2+ binding strength is observed, to an apparent dissociation constant of 760 nM. Such a strong dependence on ionic strength is not surprising for the reaction of a tetravalent anion with a divalent cation.
- the stilbene-type indicators disclosed herein can exist in cis and trans isomers. Both isomers are included in the products (e.g., IX, XII, and XV) of the Wittig reactions. Separation of the isomers was attempted only with stil-1/Me and Et. Thin layer chromatography (chloroform-methanol 9:1 or 19:1 v/v) on AgNO3-impregnated silica was barely able to resolve the two isomers of stil-1/Et, the cis isomer migrating more slowly.
- trans stil-1 Assuming only the trans isomer is fluorescent (see reference 25), the photostationary state in the absence of Ca 2+ contains about 75% trans, 25% cis. This ratio of trans to cis is unusually high for a stilbene photostationary state. If crude stil-1/Me containing the original mixture of cis and trans isomers is hydrolyzed, irradiation of the anion mixture increases the absorbance and fluorescence. Photoisomerization significantly complicates the use of trans stil-1 as a fluorescent indicator.
- stil-1 It becomes necessary to irradiate stil-1 strongly to reach photostationary equilibrium before Ca 2+ measurement begins, and even then to minimize the intensity of the excitation beam and read Ca 2+ from the ratio of excitation efficiencies at two wavelengths rather than from the absolute fluorescence intensity at any one excitation wavelength.
- stil-1 also undergoes yet another, albeit slow, photochemical reaction in addition to cis-trans isomerism. This slow reaction causes a gradual rise in fluorescence at long (>300 nm) excitation wavelengths. It is most noticeable when a stil-1 sample is irradiated at very low [Ca 2+ ], then exposed to high Ca 2+ during measurement of its excitation spectrum.
- Fura-2 is also better than quin2 in rejecting Mn 2+ , the Mn 2+ :Ca 2+ preferences being 42-fold and 510-fold respectively (see reference 26).
- the predominant oxidation state of labile intracellular iron is +2 (see reference 27); Fe 2+ binds fura-2 between three and ten time as strongly as Ca 2+ does. A more precise estimate was prevented by experimental scatter, which may reflect more complicated stoichiometry than 1:1 or imperfectly anaerobic conditions.
- Both Mn 2+ and Fe 2+ quench fura-2 as expected for metals with many unpaired electrons.
- EGTA prefers Zn 2+ , Mn 2+ , and Fe 2+ to Ca 2+ , by factors of ca. 50, ca. 20, and ca. 8-fold respectively (see reference 11).
- FIG. 7 indicates that pH variations over a reasonable range of intracellular values hardly effect either the spectra of the Ca 2+ -free or Ca 2+ -bound species or the effective Ca 2+ dissociation constant.
- Red cells were used as model membranes to test binding because they are easy to add and remove. Red cells added to a hematocrit of 17% in a solution of fura-2, fura-3, or indo-1, then centrifuged out, decreased the fluorescence of the supernatant by less than 5%. This small drop in fluorescence could have been just dilution by the small unavoidable amount of extracellular fluid added with the packed cells. Even if it were attributed entirely to binding, the amount of dye bound to a membrane would be less than that contained in a layer 0.2 microns thick of adjacent aqueous solution. Such negligible binding was found whether the medium contained 1 mM Ca 2+ or EGTA without Ca 2+ .
- D A and D B are related to [Ca 2+ ]by the equation for 1:1 complexation
- Equation 2 a form closely analogous to the calibration equation for a dye using intensity values at only one wavelength: ##EQU4##
- equations (5) and (6) assume that the dye forms a simple 1:1 complex with Ca 2+ (equation 2), that it behaves in cells as it does in calibration media and that it is sufficiently dilute for fluorescence intensity to be linearly proportional to the concentrations of the fluorescent species (equation 1).
- equation 6 requires that F, F min , and F max all be determined at the same instrumental sensitivity, optical path length, and effective total concentration of dye.
- ratio operation has two main disadvantages. If tissue autofluorescence is significant, it has to be subtracted before forming the ratio, whereas it cancels out automatically in equation 6 as long as it is calcium-independent. Fortunately the brightness of the new dyes makes it easy to swamp autofluorescence. Also the instrumentation to alternate excitation wavelengths rapidly and to calculate the ratio of fluorescence intensities is not yet standardly available. In our laboratory at the University of California we have built a system in which two light sources and two monochromators are alternately selected by a butterfly-shaped sector mirror rotating at 10-30 Hz. Cancellation of large fluctuations in intensity has indeed been invaluable in studying monolayers and single small cells. However, it would be relatively difficult to provide sequential excitation wavelengths of stable relative intensity in flow cytometry, so that the emission shift of indo-1 makes it particularly attractive in this application.
- the present invention discloses a new class of fluorescent, indicator dyes that are specific for calcium ions.
- the new fluorescent, indicator dyes combine a stilbene-type fluorophore with a tetracarboxylate parent Ca 2+ chelating compound having the octacoordinate pattern of ligating groups characteristic of EGTA and BAPTA.
- the new dyes are composed of a BAPTA-like chelator coupled to a single fluorescent dye.
- the new dyes are comprised of a BAPTA-like chelator coupled to a pair of fluorescent dyes.
- Preferred compounds contain heterocyclic brides to reinforce the ethylenic bond of the stilbene and to reduce hydrophobicity.
- the new dye compounds disclosed herein are superior to existing fluorescent calcium indicators such as quin2 because the new compounds show a much stronger fluorescence, a somewhat weaker affinity for Ca 2+ , a better selectivity against magnesium and heavy metals, and most importantly a wavelength shift upon Ca 2+ -binding.
- the new dye indicators should not only supersede quin2 in many applications, but should also ease [Ca 2+ ] i measurements in adherent cells, bulk tissues, or single cells under a microscope or in a flow cytometer.
- the present invention discloses an improved method for synthesizing alpha-acyloxyalkyl bromides wherein the bromides wherein the bromides so synthesized are free of contaminating bis(1-bromoalkyl)ether.
- alpha-acyloxy bromides are useful to the pharmaceutical industry in the preparation of prodrugs (see references 38 and 39).
- the improved method is specifically exemplified herein in the synthesis of acetoxymethyl bromide, a compound useful in preparing the acetoxymethyl esters disclosed and claimed herein.
Abstract
Description
I.sub.1 =D.sub.A F.sub.A1 +D.sub.B F.sub.B1 (1a )
I.sub.2 =D.sub.A F.sub.A2 +D.sub.B F.sub.B2 (1b)
D.sub.B =D.sub.A [Ca.sup.2+ ]/K.sub.d (2)
TABLE 1 __________________________________________________________________________ PROPERTIES OF NEW FLUORESCENT INDICATORS OF Ca.sup.2+ Apparent K.sub.d Fluorescence for Ca.sup.2+ Absorption maxima Emission maxima quantum efficiency DYE (nM) Free anion Ca complex Free anion Ca complex Free anion Ca complex __________________________________________________________________________ stil-1 132.sup.a, 200.sup.b 362(27) 329(34) 537(585) 529(n.d.) .14 n.d. stil-2 224.sup.c 352(12) 326(12) 564(590) 560(587) .11 .15 indo-1 250.sup.b 349(34) 331(34) 485(482) 410(398) .38 .56 fura-1 107.sup.b 350(21) 334(27) 534(585) 522(548) .10 .20 fura-2 135.sup.a, 224.sup.b 362(27) 335(33) 512(518) 505(510) .23 .49 fura-3 140.sup.b 370(14) 343(25) 564(588) 551(599) .13 .21 quin-2.sup.d 80.sup.a 354(5) 332(5) 492(510) 498(525) .03 .14 __________________________________________________________________________ .sup.a In 100 mM KCl, 20° C., pH 7.1-7.2 .sup.b In 115 mM KCl, 20 mM NaCl, 10 mM KMOPS, pH 7.05, 1 mM free Mg.sup.2+, 37° C. .sup.c In 100 mM KCl, 37° C., pH 7.08 .sup.d Shown here for comparison. Data taken fromreference 4, p. 106. n.d., not determined Absorption maxima refer to the dominant peaks at longest wavelength, measured at 22 ± 2° C. in 100 mM KCl. The first number is the wavelength in nm, followed in parentheses by 10.sup.-3 × the corresponding extinction coefficient, M.sup.-1 cm.sup.-1. The data for stil2 are for the cistrans mixture as saponified; both the wavelengths an extinction coefficients would increase for pure trans. Extinction coefficients for fura1 and fura3 may be underestimated since less effort was expended on their purification than on stil1, indo1, and fura2. Emission maxima list the peak wavelengths in nm of the uncorrected and corrected (in parentheses) emission spectra. The differences between uncorrected and corrected values are greatest for stil1 and fura1 because they were measured on the PerkinElmer MPF44, whose emission characteristics are more biased than the Spex Fluorolog, on which the others were determined. Emission maxima and quantum efficiencies were measured in 100 mM KCl, 22 ± 2° C.
Claims (30)
Priority Applications (6)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US06/648,649 US4603209A (en) | 1984-09-07 | 1984-09-07 | Fluorescent indicator dyes for calcium ions |
CA000489749A CA1254896A (en) | 1984-09-07 | 1985-08-30 | Fluorescent indicator dyes for calcium ions |
JP60197531A JPH0694444B2 (en) | 1984-09-07 | 1985-09-06 | Calcium specific fluorescent indicator dye |
DE8585306387T DE3566112D1 (en) | 1984-09-07 | 1985-09-09 | Fluorescent indicator dyes for calcium ions |
EP85306387A EP0178775B1 (en) | 1984-09-07 | 1985-09-09 | Fluorescent indicator dyes for calcium ions |
AT85306387T ATE38509T1 (en) | 1984-09-07 | 1985-09-09 | FLUORESCENT INDICATOR DYES FOR CALCIUM IONS. |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US06/648,649 US4603209A (en) | 1984-09-07 | 1984-09-07 | Fluorescent indicator dyes for calcium ions |
Publications (1)
Publication Number | Publication Date |
---|---|
US4603209A true US4603209A (en) | 1986-07-29 |
Family
ID=24601639
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US06/648,649 Expired - Lifetime US4603209A (en) | 1984-09-07 | 1984-09-07 | Fluorescent indicator dyes for calcium ions |
Country Status (6)
Country | Link |
---|---|
US (1) | US4603209A (en) |
EP (1) | EP0178775B1 (en) |
JP (1) | JPH0694444B2 (en) |
AT (1) | ATE38509T1 (en) |
CA (1) | CA1254896A (en) |
DE (1) | DE3566112D1 (en) |
Cited By (150)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US4761481A (en) * | 1985-03-18 | 1988-08-02 | Baxter Travenol Laboratories, Inc. | Substituted pyridine derivatives |
US4795712A (en) * | 1987-04-10 | 1989-01-03 | Eastman Kodak Company | Calcium complexing dyes and their use in analytical compositions, elements and methods |
US4806604A (en) * | 1987-05-13 | 1989-02-21 | Regents Of The University Of California | Photosensitive calcium chelators |
US5049673A (en) * | 1987-10-30 | 1991-09-17 | The Regents Of The University Of California | Fluorescent indicator dyes for calcium working at long wavelengths |
US5141627A (en) * | 1989-09-25 | 1992-08-25 | The Regents Of The University Of California | Chelators whose affinity for calcium ion is increased by illumination |
US5262330A (en) * | 1992-02-27 | 1993-11-16 | Miles Inc. | Colorimetric methods and reagents for the assay of calcium in a test sample |
US5294799A (en) * | 1993-02-01 | 1994-03-15 | Aslund Nils R D | Apparatus for quantitative imaging of multiple fluorophores |
US5310888A (en) * | 1992-10-22 | 1994-05-10 | Miles Inc. | Arylazo chromoionophores |
WO1994010160A1 (en) * | 1992-10-23 | 1994-05-11 | The Regents Of The University Of California | Novel azide-containing chelators whose affinity for calcium ion is decreased by illumination |
US5319209A (en) * | 1991-11-20 | 1994-06-07 | Hammamatsu Photonics K.K. | Method for measurement of intracellular ion concentration using fluorescence probe dyes |
US5324747A (en) * | 1992-07-15 | 1994-06-28 | Hoffmann-La Roche Inc. | N-substituted anilines, inhibitors of phospholipases A2 |
US5358844A (en) * | 1993-02-18 | 1994-10-25 | Brigham And Women's Hospital, Inc. | Preservation of blood platelets |
US5405975A (en) * | 1993-03-29 | 1995-04-11 | Molecular Probes, Inc. | Fluorescent ion-selective diaryldiaza crown ether conjugates |
US5409835A (en) * | 1992-12-30 | 1995-04-25 | The University Of Maryland At Baltimore | Long-wavelength fluorescent probe compounds for calcium ions and their use in ratiometrically measuring calcium ion concentrations |
US5453517A (en) * | 1992-02-25 | 1995-09-26 | Molecular Probes, Inc. | Reactive derivatives of bapta used to make ion-selective chelators |
US5459276A (en) * | 1994-05-20 | 1995-10-17 | Molecular Probes, Inc. | Benzazolylcoumarin-based ion indicators for heavy metals |
US5462989A (en) * | 1990-11-07 | 1995-10-31 | Mauze; Ganapati R. | Fiber optic potassium ion sensor |
US5501980A (en) * | 1994-05-20 | 1996-03-26 | Molecular Probes, Inc. | Benzazolylcoumarin-based ion indicators |
US5516911A (en) * | 1993-12-30 | 1996-05-14 | The United States Of America As Represented By The Department Of Health And Human Services | Fluorescent intracellular calcium indicators |
US5550031A (en) * | 1992-08-24 | 1996-08-27 | Hamamatsu Photonics K.K. | Method of optimizing equilibrium constants and fluorescence coefficients for measuring intracellular ion concentration using fluorescence probe dyes with triple wave length photometry |
US5576433A (en) * | 1994-10-12 | 1996-11-19 | Texas Fluorescence Laboratories, Inc. | Fluorescent calcium indicators targeted to specific intracellular environments |
US5599831A (en) * | 1994-05-27 | 1997-02-04 | Poretz; Ronald D. | Method of preparation of pharmaceutical compositions |
US5648270A (en) * | 1995-02-06 | 1997-07-15 | Molecular Probes, Inc. | Methods of sensing with fluorescent conjugates of metal-chelating nitrogen heterocycles |
US5747345A (en) * | 1996-08-15 | 1998-05-05 | Johnson & Johnson Clinical Diagnostics, Inc. | Calcium specific diaza-18-crown-6-ether chromoionophores |
US5773227A (en) * | 1993-06-23 | 1998-06-30 | Molecular Probes, Inc. | Bifunctional chelating polysaccharides |
US6013764A (en) * | 1996-07-17 | 2000-01-11 | Ortho Pharmaceutical Corp. | Liquid phase peptide synthesis of KL-4 pulmonary surfactant |
US6171866B1 (en) | 1998-09-30 | 2001-01-09 | Avl Medical Instruments | Luminescence indicator for determining calcium ions |
US6180411B1 (en) | 1997-08-01 | 2001-01-30 | The Regents Of The University Of California | Light-triggered indicators that memorize analyte concentrations |
DE19915137C2 (en) * | 1999-03-26 | 2001-10-18 | Michael Schaefer | Fluorescence microscopy method for quantifying multiple fluorochromes in a multi-colored sample and uses of the method |
US6469154B1 (en) | 1999-05-21 | 2002-10-22 | The Regents Of The University Of California | Fluorescent protein indicators |
US20020157120A1 (en) * | 1999-05-21 | 2002-10-24 | Tsien Roger Y. | Circularly permuted fluorescent protein indicators |
US20030073149A1 (en) * | 2001-10-12 | 2003-04-17 | Archer Robert A. | Antibody complexes and methods for immunolabeling |
US6670449B1 (en) | 1997-02-13 | 2003-12-30 | Memorial Sloan-Kettering Cancer Center | Hybrid molecules and their use for optically detecting changes in cellular microenvironments |
US20040038306A1 (en) * | 2002-05-03 | 2004-02-26 | Brian Agnew | Compositions and methods for detection and isolation of phosphorylated molecules |
US6699687B1 (en) | 1999-05-21 | 2004-03-02 | The Regents Of The University Of California | Circularly permuted fluorescent protein indicators |
US20040147747A1 (en) * | 2000-08-04 | 2004-07-29 | Molecular Probes, Inc. | Derivatives of 1,2-dihydro-7-hydroxyquinolines containing fused rings |
US20040171034A1 (en) * | 2002-05-03 | 2004-09-02 | Brian Agnew | Compositions and methods for detection and isolation of phosphorylated molecules |
US20050014197A1 (en) * | 2002-05-03 | 2005-01-20 | Brian Agnew | Compositions and methods for detection and isolation of phosphorylated molecules |
US20050064485A1 (en) * | 2003-09-12 | 2005-03-24 | Kurt Vogel | Multiplex binding and activity assays |
US20050181464A1 (en) * | 2002-04-04 | 2005-08-18 | Affinium Pharmaceuticals, Inc. | Novel purified polypeptides from bacteria |
US20050191643A1 (en) * | 2003-08-11 | 2005-09-01 | Rosaria Haugland | Cyanine compounds and their application as quenching compounds |
US20050208534A1 (en) * | 2003-12-05 | 2005-09-22 | Dallwig Jason A | Methine-substituted cyanine dye compounds |
US20050233467A1 (en) * | 2004-04-15 | 2005-10-20 | Akwasi Minta | Visible wavelength fluorescent calcium indicators that are (i) leakage resistant and (ii) operate near membranes |
US6962992B2 (en) | 2000-12-20 | 2005-11-08 | Molecullar Probes, Inc. | Crown ether derivatives |
US20050250214A1 (en) * | 2004-05-05 | 2005-11-10 | Gee Kyle R | Zinc binding compounds and their method of use |
WO2006023231A2 (en) * | 2004-07-27 | 2006-03-02 | Molecular Probes, Inc. | Fluorescent metal ion indicators with large stokes shift |
US20060160068A1 (en) * | 2003-09-17 | 2006-07-20 | Joseph Beechem | Competitive immunoassay |
DE102005022880A1 (en) * | 2005-05-18 | 2006-11-30 | Olympus Soft Imaging Solutions Gmbh | Separation of spectrally or color superimposed image contributions in a multi-color image, especially in transmission microscopic multi-color images |
US20070043212A1 (en) * | 2000-12-20 | 2007-02-22 | Molecular Probes, Inc. | Crown ether derivatives |
US20070154958A1 (en) * | 2005-10-13 | 2007-07-05 | Aureon Laboratories, Inc. | Multiplex in situ immunohistochemical analysis |
US20070161112A1 (en) * | 2006-01-12 | 2007-07-12 | Invitrogen Corporation | Heavy metal binding compounds and their method of use |
US20070190597A1 (en) * | 2006-02-10 | 2007-08-16 | Invitrogen Corporation | Oligosaccharide modification and labeling of proteins |
US20070224652A1 (en) * | 2006-03-23 | 2007-09-27 | Jan Holgersson | Blood Group Antigens of Different Types for Diagnostic and Therapeutic Applications |
WO2007124464A2 (en) | 2006-04-20 | 2007-11-01 | Becton, Dickinson And Company | Thermostable proteins and methods of making and using thereof |
US20080009026A1 (en) * | 2006-07-07 | 2008-01-10 | Invitrogen Corporation | Fluorogenic protein kinase substrates |
US20080160627A1 (en) * | 2003-11-19 | 2008-07-03 | Invitrogen Corporation | Environmental Sensitive Fluorogenic Compounds and Their Application for Singlet Oxygen and Protein Detection |
US20080254498A1 (en) * | 2007-04-13 | 2008-10-16 | Abd Bioquest, Inc. | Fluorescent ion indicators and their applications |
US7446202B2 (en) | 2003-12-05 | 2008-11-04 | Molecular Probes, Inc. | Cyanine dye compounds |
US7465848B2 (en) | 2002-11-20 | 2008-12-16 | The General Hospital Corporation | Zebrafish assay |
US20090004753A1 (en) * | 2007-01-30 | 2009-01-01 | Invitrogen Corporation | Labeling reagents and methods of their use |
WO2009031734A1 (en) * | 2007-09-07 | 2009-03-12 | Korea University Industrial & Academic Collaboration Foundation | Two-photon probe for real-time monitoring of intracellular calcium ions, method for preparing the probe and method for real-time monitoring of intracellular calcium ions using the probe |
US20090081722A1 (en) * | 2002-09-12 | 2009-03-26 | Invitrogen Corporation | Site-specific labeling of affinity tags in fusion proteins |
US7582461B2 (en) | 2003-07-29 | 2009-09-01 | Life Technologies Corporation | Kinase and phosphatase assays |
US7598390B2 (en) | 2005-05-11 | 2009-10-06 | Life Technologies Corporation | Fluorescent chemical compounds having high selectivity for double stranded DNA, and methods for their use |
US7619059B2 (en) | 2003-07-29 | 2009-11-17 | Life Technologies Corporation | Bimolecular optical probes |
US20100009342A1 (en) * | 2005-09-06 | 2010-01-14 | Life Technologies Corporation | Control of chemical modification |
US7727752B2 (en) | 2003-07-29 | 2010-06-01 | Life Technologies Corporation | Kinase and phosphatase assays |
US20100167320A1 (en) * | 2006-01-30 | 2010-07-01 | Life Technologies Corporation | Compositions and Methods for Detecting and Quantifying Toxic Substances in Disease States |
US20100203564A1 (en) * | 2007-05-30 | 2010-08-12 | Life Technologies Corporation | Fluorescent Phospholipase A2 Indicators |
EP2224013A1 (en) | 2007-02-13 | 2010-09-01 | Life Technologies Corporation | Methods for the determination of telomerase activity using click chemistry |
WO2010099137A2 (en) | 2009-02-26 | 2010-09-02 | Osi Pharmaceuticals, Inc. | In situ methods for monitoring the emt status of tumor cells in vivo |
US20100240071A1 (en) * | 2007-06-19 | 2010-09-23 | Rajiv Gandhi Centre For Biotechnology | Assay for Detection of Transient Intracellular CA2+ |
US20100255601A1 (en) * | 2006-01-24 | 2010-10-07 | Invitrogen Corporation | Device and methods for quantifying analytes |
US7875047B2 (en) | 2002-04-19 | 2011-01-25 | Pelikan Technologies, Inc. | Method and apparatus for a multi-use body fluid sampling device with sterility barrier release |
US7892183B2 (en) | 2002-04-19 | 2011-02-22 | Pelikan Technologies, Inc. | Method and apparatus for body fluid sampling and analyte sensing |
US7901365B2 (en) | 2002-04-19 | 2011-03-08 | Pelikan Technologies, Inc. | Method and apparatus for penetrating tissue |
US7909778B2 (en) | 2002-04-19 | 2011-03-22 | Pelikan Technologies, Inc. | Method and apparatus for penetrating tissue |
US7909777B2 (en) | 2002-04-19 | 2011-03-22 | Pelikan Technologies, Inc | Method and apparatus for penetrating tissue |
US7909774B2 (en) | 2002-04-19 | 2011-03-22 | Pelikan Technologies, Inc. | Method and apparatus for penetrating tissue |
US7909775B2 (en) | 2001-06-12 | 2011-03-22 | Pelikan Technologies, Inc. | Method and apparatus for lancet launching device integrated onto a blood-sampling cartridge |
US7914465B2 (en) | 2002-04-19 | 2011-03-29 | Pelikan Technologies, Inc. | Method and apparatus for penetrating tissue |
WO2011047391A1 (en) | 2009-10-17 | 2011-04-21 | Asante Research, Llc | Fluorescent calcium indicators that are ratiometric and emit in the red spectrum |
US20110118142A1 (en) * | 2008-05-16 | 2011-05-19 | Life Technologies Corporation | Dual labeling methods for measuring cellular proliferation |
US20110159517A1 (en) * | 2003-05-05 | 2011-06-30 | Life Technologies Corporation | Zinc binding compounds and their method of use |
US7976476B2 (en) | 2002-04-19 | 2011-07-12 | Pelikan Technologies, Inc. | Device and method for variable speed lancet |
US7981055B2 (en) | 2001-06-12 | 2011-07-19 | Pelikan Technologies, Inc. | Tissue penetration device |
US7981056B2 (en) | 2002-04-19 | 2011-07-19 | Pelikan Technologies, Inc. | Methods and apparatus for lancet actuation |
US7988645B2 (en) | 2001-06-12 | 2011-08-02 | Pelikan Technologies, Inc. | Self optimizing lancing device with adaptation means to temporal variations in cutaneous properties |
US8007446B2 (en) | 2002-04-19 | 2011-08-30 | Pelikan Technologies, Inc. | Method and apparatus for penetrating tissue |
US8062231B2 (en) | 2002-04-19 | 2011-11-22 | Pelikan Technologies, Inc. | Method and apparatus for penetrating tissue |
US8079960B2 (en) | 2002-04-19 | 2011-12-20 | Pelikan Technologies, Inc. | Methods and apparatus for lancet actuation |
WO2012022734A2 (en) | 2010-08-16 | 2012-02-23 | Medimmune Limited | Anti-icam-1 antibodies and methods of use |
US8197421B2 (en) | 2002-04-19 | 2012-06-12 | Pelikan Technologies, Inc. | Method and apparatus for penetrating tissue |
US8221334B2 (en) | 2002-04-19 | 2012-07-17 | Sanofi-Aventis Deutschland Gmbh | Method and apparatus for penetrating tissue |
US8251921B2 (en) | 2003-06-06 | 2012-08-28 | Sanofi-Aventis Deutschland Gmbh | Method and apparatus for body fluid sampling and analyte sensing |
US8262614B2 (en) | 2003-05-30 | 2012-09-11 | Pelikan Technologies, Inc. | Method and apparatus for fluid injection |
US8267870B2 (en) | 2002-04-19 | 2012-09-18 | Sanofi-Aventis Deutschland Gmbh | Method and apparatus for body fluid sampling with hybrid actuation |
US8282576B2 (en) | 2003-09-29 | 2012-10-09 | Sanofi-Aventis Deutschland Gmbh | Method and apparatus for an improved sample capture device |
US8296918B2 (en) | 2003-12-31 | 2012-10-30 | Sanofi-Aventis Deutschland Gmbh | Method of manufacturing a fluid sampling device with improved analyte detecting member configuration |
US8318450B2 (en) | 2004-01-21 | 2012-11-27 | Life Technologies Corporation | Optically-detectable enzyme substrates and their method of use |
US8333710B2 (en) | 2002-04-19 | 2012-12-18 | Sanofi-Aventis Deutschland Gmbh | Tissue penetration device |
US8360992B2 (en) | 2002-04-19 | 2013-01-29 | Sanofi-Aventis Deutschland Gmbh | Method and apparatus for penetrating tissue |
US8372016B2 (en) | 2002-04-19 | 2013-02-12 | Sanofi-Aventis Deutschland Gmbh | Method and apparatus for body fluid sampling and analyte sensing |
US8382682B2 (en) | 2002-04-19 | 2013-02-26 | Sanofi-Aventis Deutschland Gmbh | Method and apparatus for penetrating tissue |
US8435190B2 (en) | 2002-04-19 | 2013-05-07 | Sanofi-Aventis Deutschland Gmbh | Method and apparatus for penetrating tissue |
US8439872B2 (en) | 1998-03-30 | 2013-05-14 | Sanofi-Aventis Deutschland Gmbh | Apparatus and method for penetration with shaft having a sensor for sensing penetration depth |
US8540521B2 (en) | 2008-12-29 | 2013-09-24 | Life Technologies Corporation | Benzoxazole-based fluorescent metal ion indicators |
US8556829B2 (en) | 2002-04-19 | 2013-10-15 | Sanofi-Aventis Deutschland Gmbh | Method and apparatus for penetrating tissue |
US8562802B1 (en) | 2006-02-13 | 2013-10-22 | Life Technologies Corporation | Transilluminator base and scanner for imaging fluorescent gels, charging devices and portable electrophoresis systems |
US8574895B2 (en) | 2002-12-30 | 2013-11-05 | Sanofi-Aventis Deutschland Gmbh | Method and apparatus using optical techniques to measure analyte levels |
US8641644B2 (en) | 2000-11-21 | 2014-02-04 | Sanofi-Aventis Deutschland Gmbh | Blood testing apparatus having a rotatable cartridge with multiple lancing elements and testing means |
US8652831B2 (en) | 2004-12-30 | 2014-02-18 | Sanofi-Aventis Deutschland Gmbh | Method and apparatus for analyte measurement test time |
US8668656B2 (en) | 2003-12-31 | 2014-03-11 | Sanofi-Aventis Deutschland Gmbh | Method and apparatus for improving fluidic flow and sample capture |
US8702624B2 (en) | 2006-09-29 | 2014-04-22 | Sanofi-Aventis Deutschland Gmbh | Analyte measurement device with a single shot actuator |
WO2014066733A2 (en) | 2012-10-25 | 2014-05-01 | Life Technologies Corporation | Methods and compositions for enzyme-mediated site-specific radiolabeling of glycoproteins |
EP2727965A1 (en) | 2006-10-27 | 2014-05-07 | Life Technologies Corporation | Fluorogenic pH sensitive dyes and their method of use |
US8721671B2 (en) | 2001-06-12 | 2014-05-13 | Sanofi-Aventis Deutschland Gmbh | Electric lancet actuator |
WO2014085160A1 (en) | 2012-11-29 | 2014-06-05 | Idexx Laboratories, Inc. | Multi-layer device for selectively determining magnesium ion |
US8784335B2 (en) | 2002-04-19 | 2014-07-22 | Sanofi-Aventis Deutschland Gmbh | Body fluid sampling device with a capacitive sensor |
US8828203B2 (en) | 2004-05-20 | 2014-09-09 | Sanofi-Aventis Deutschland Gmbh | Printable hydrogels for biosensors |
US8965476B2 (en) | 2010-04-16 | 2015-02-24 | Sanofi-Aventis Deutschland Gmbh | Tissue penetration device |
US8974651B2 (en) | 2010-04-17 | 2015-03-10 | C.C. Imex | Illuminator for visualization of fluorophores |
WO2015050959A1 (en) | 2013-10-01 | 2015-04-09 | Yale University | Anti-kit antibodies and methods of use thereof |
US9097730B2 (en) | 2007-04-13 | 2015-08-04 | Aat Bioquest, Inc. | Fluorescein lactone ion indicators and their applications |
US9144401B2 (en) | 2003-06-11 | 2015-09-29 | Sanofi-Aventis Deutschland Gmbh | Low pain penetrating member |
US9226699B2 (en) | 2002-04-19 | 2016-01-05 | Sanofi-Aventis Deutschland Gmbh | Body fluid sampling module with a continuous compression tissue interface surface |
US9248267B2 (en) | 2002-04-19 | 2016-02-02 | Sanofi-Aventis Deustchland Gmbh | Tissue penetration device |
US9279817B2 (en) | 2007-04-13 | 2016-03-08 | Aat Bioquest, Inc. | Carbofluorescein lactone ion indicators and their applications |
US9314194B2 (en) | 2002-04-19 | 2016-04-19 | Sanofi-Aventis Deutschland Gmbh | Tissue penetration device |
US9351680B2 (en) | 2003-10-14 | 2016-05-31 | Sanofi-Aventis Deutschland Gmbh | Method and apparatus for a variable user interface |
US9375169B2 (en) | 2009-01-30 | 2016-06-28 | Sanofi-Aventis Deutschland Gmbh | Cam drive for managing disposable penetrating member actions with a single motor and motor and control system |
US9386944B2 (en) | 2008-04-11 | 2016-07-12 | Sanofi-Aventis Deutschland Gmbh | Method and apparatus for analyte detecting device |
WO2016111915A1 (en) | 2015-01-06 | 2016-07-14 | De Haas Anthony H | Near-infrared fluorescent surgical dye markers |
US9427532B2 (en) | 2001-06-12 | 2016-08-30 | Sanofi-Aventis Deutschland Gmbh | Tissue penetration device |
US9504405B2 (en) | 2013-10-23 | 2016-11-29 | Verily Life Sciences Llc | Spatial modulation of magnetic particles in vasculature by external magnetic field |
US9775553B2 (en) | 2004-06-03 | 2017-10-03 | Sanofi-Aventis Deutschland Gmbh | Method and apparatus for a fluid sampling device |
WO2017181176A1 (en) | 2016-04-15 | 2017-10-19 | Lcahn School Of Medicine At Mount Sinai | Tissue profiling using multiplexed immunohistochemical consecutive staining |
US9795747B2 (en) | 2010-06-02 | 2017-10-24 | Sanofi-Aventis Deutschland Gmbh | Methods and apparatus for lancet actuation |
US9810700B1 (en) | 2017-05-31 | 2017-11-07 | Aat Bioquest, Inc. | Fluorogenic calcium ion indicators and methods of using the same |
US9820684B2 (en) | 2004-06-03 | 2017-11-21 | Sanofi-Aventis Deutschland Gmbh | Method and apparatus for a fluid sampling device |
US9835587B2 (en) | 2014-04-01 | 2017-12-05 | C.C. Imex | Electrophoresis running tank assembly |
US9861710B1 (en) | 2015-01-16 | 2018-01-09 | Verily Life Sciences Llc | Composite particles, methods, and in vivo diagnostic system |
US10028659B2 (en) | 2015-03-26 | 2018-07-24 | Verily Life Sciences Llc | Aptamer-based sensors, implantable devices and detection system |
WO2018183621A1 (en) | 2017-03-30 | 2018-10-04 | Life Technologies Corporation | Quantification of ngs dna by adapter sequence |
US10542918B2 (en) | 2013-10-23 | 2020-01-28 | Verily Life Sciences Llc | Modulation of a response signal to distinguish between analyte and background signals |
CN111056985A (en) * | 2020-01-02 | 2020-04-24 | 广州大学 | Partially cyanine derivative fluorescent probe and preparation method and application thereof |
CN111138387A (en) * | 2020-01-02 | 2020-05-12 | 广州大学 | Cyano furanone derivative fluorescent probe and preparation method and application thereof |
EP3733244A1 (en) | 2013-10-02 | 2020-11-04 | Medlmmune, LLC | Neutralizing anti-influenza a antibodies and uses thereof |
WO2022006368A2 (en) | 2020-07-02 | 2022-01-06 | Life Technologies Corporation | Trinucleotide cap analogs, preparation and uses thereof |
WO2023004400A1 (en) | 2021-07-21 | 2023-01-26 | Life Technologies Corporation | Dibenzoxanthene quenchers, uses, and methods of preparation |
Families Citing this family (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
AU621858B2 (en) * | 1988-03-28 | 1992-03-26 | Abbott Laboratories | Calcium assay reagent |
JPH01267443A (en) * | 1988-04-19 | 1989-10-25 | Mitsubishi Kasei Corp | Method and apparatus for optical measurement |
US5958782A (en) * | 1993-10-21 | 1999-09-28 | Minnesota Mining And Manufacturing Company | Cation-sensing composite structure and compounds for use therein |
ATE260466T1 (en) * | 1997-03-21 | 2004-03-15 | Biotechnolog Forschung Gmbh | COMPOUNDS FOR DETECTING PHOSPHORIC ACID ESTERS |
Family Cites Families (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CA1007551A (en) * | 1973-04-11 | 1977-03-29 | The Dow Chemical Company | Determination of calcium |
-
1984
- 1984-09-07 US US06/648,649 patent/US4603209A/en not_active Expired - Lifetime
-
1985
- 1985-08-30 CA CA000489749A patent/CA1254896A/en not_active Expired
- 1985-09-06 JP JP60197531A patent/JPH0694444B2/en not_active Expired - Lifetime
- 1985-09-09 AT AT85306387T patent/ATE38509T1/en not_active IP Right Cessation
- 1985-09-09 DE DE8585306387T patent/DE3566112D1/en not_active Expired
- 1985-09-09 EP EP85306387A patent/EP0178775B1/en not_active Expired
Non-Patent Citations (6)
Title |
---|
Arthur H. Schmidt, "Bromotrimethylsilane--Versatile Reagents for Organic Synthesis", Aldrichimica Acta., vol. 14, No. 2, 1981. |
Arthur H. Schmidt, Bromotrimethylsilane Versatile Reagents for Organic Synthesis , Aldrichimica Acta., vol. 14, No. 2, 1981. * |
Roger Y. Tsien "Intracellular Measurements of Ion Activities" Ann. Rev. Biophys. Bioeng., 1983, 12:91-116. |
Roger Y. Tsien Intracellular Measurements of Ion Activities Ann. Rev. Biophys. Bioeng., 1983, 12:91 116. * |
Roger Y. Tsien, "New Calcium Indicators and Buffers with High Selectivity Against Magnesium and Protons: Design, Synthesis, and Properties of Prototype", Am. Chem. Soc., 1980. |
Roger Y. Tsien, New Calcium Indicators and Buffers with High Selectivity Against Magnesium and Protons: Design, Synthesis, and Properties of Prototype , Am. Chem. Soc., 1980. * |
Cited By (324)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US4761481A (en) * | 1985-03-18 | 1988-08-02 | Baxter Travenol Laboratories, Inc. | Substituted pyridine derivatives |
US4795712A (en) * | 1987-04-10 | 1989-01-03 | Eastman Kodak Company | Calcium complexing dyes and their use in analytical compositions, elements and methods |
US4806604A (en) * | 1987-05-13 | 1989-02-21 | Regents Of The University Of California | Photosensitive calcium chelators |
US5049673A (en) * | 1987-10-30 | 1991-09-17 | The Regents Of The University Of California | Fluorescent indicator dyes for calcium working at long wavelengths |
US5141627A (en) * | 1989-09-25 | 1992-08-25 | The Regents Of The University Of California | Chelators whose affinity for calcium ion is increased by illumination |
US5462989A (en) * | 1990-11-07 | 1995-10-31 | Mauze; Ganapati R. | Fiber optic potassium ion sensor |
US5319209A (en) * | 1991-11-20 | 1994-06-07 | Hammamatsu Photonics K.K. | Method for measurement of intracellular ion concentration using fluorescence probe dyes |
US5453517A (en) * | 1992-02-25 | 1995-09-26 | Molecular Probes, Inc. | Reactive derivatives of bapta used to make ion-selective chelators |
US5262330A (en) * | 1992-02-27 | 1993-11-16 | Miles Inc. | Colorimetric methods and reagents for the assay of calcium in a test sample |
US5446189A (en) * | 1992-07-15 | 1995-08-29 | Hoffmann-La Roche Inc. | Intermediates for the production of N-substituted anilines, inhibitors of phospholipases A2 |
AU666199B2 (en) * | 1992-07-15 | 1996-02-01 | F. Hoffmann-La Roche Ag | N-substituted anilines |
US5324747A (en) * | 1992-07-15 | 1994-06-28 | Hoffmann-La Roche Inc. | N-substituted anilines, inhibitors of phospholipases A2 |
US5550031A (en) * | 1992-08-24 | 1996-08-27 | Hamamatsu Photonics K.K. | Method of optimizing equilibrium constants and fluorescence coefficients for measuring intracellular ion concentration using fluorescence probe dyes with triple wave length photometry |
US5866355A (en) * | 1992-08-24 | 1999-02-02 | Hamamatsu Photonics, K.K. | Device for measuring an intracellular ion concentration |
US5310888A (en) * | 1992-10-22 | 1994-05-10 | Miles Inc. | Arylazo chromoionophores |
US5552555A (en) * | 1992-10-23 | 1996-09-03 | The Regents Of University Of California | Azide-containing chelators whose affinity for calcium ion is decreased by illumination |
WO1994010160A1 (en) * | 1992-10-23 | 1994-05-11 | The Regents Of The University Of California | Novel azide-containing chelators whose affinity for calcium ion is decreased by illumination |
US5602258A (en) * | 1992-10-23 | 1997-02-11 | The Regents Of University Of California | Azide-containing chelators whose affinity for calcium ion is decreased by illumination |
US5409835A (en) * | 1992-12-30 | 1995-04-25 | The University Of Maryland At Baltimore | Long-wavelength fluorescent probe compounds for calcium ions and their use in ratiometrically measuring calcium ion concentrations |
US5294799A (en) * | 1993-02-01 | 1994-03-15 | Aslund Nils R D | Apparatus for quantitative imaging of multiple fluorophores |
US5876676A (en) * | 1993-02-18 | 1999-03-02 | Brigham And Women's Hospital, Inc. | Preservation of blood platelets |
US5358844A (en) * | 1993-02-18 | 1994-10-25 | Brigham And Women's Hospital, Inc. | Preservation of blood platelets |
US5576213A (en) * | 1993-02-18 | 1996-11-19 | Brigham & Women's Hospital, Inc. | Preservation of blood platelets |
US5516864A (en) * | 1993-03-29 | 1996-05-14 | Molecular Probes, Inc. | Fluorescent ion-selective diaryldiaza crown ether conjugates |
US5405975A (en) * | 1993-03-29 | 1995-04-11 | Molecular Probes, Inc. | Fluorescent ion-selective diaryldiaza crown ether conjugates |
US5773227A (en) * | 1993-06-23 | 1998-06-30 | Molecular Probes, Inc. | Bifunctional chelating polysaccharides |
US5516911A (en) * | 1993-12-30 | 1996-05-14 | The United States Of America As Represented By The Department Of Health And Human Services | Fluorescent intracellular calcium indicators |
US5459276A (en) * | 1994-05-20 | 1995-10-17 | Molecular Probes, Inc. | Benzazolylcoumarin-based ion indicators for heavy metals |
US5501980A (en) * | 1994-05-20 | 1996-03-26 | Molecular Probes, Inc. | Benzazolylcoumarin-based ion indicators |
US5599831A (en) * | 1994-05-27 | 1997-02-04 | Poretz; Ronald D. | Method of preparation of pharmaceutical compositions |
US5576433A (en) * | 1994-10-12 | 1996-11-19 | Texas Fluorescence Laboratories, Inc. | Fluorescent calcium indicators targeted to specific intracellular environments |
US6013802A (en) * | 1995-02-06 | 2000-01-11 | Molecular Probes, Inc. | Fluorescent conjugates of metal-chelating nitrogen heterocycles |
US5648270A (en) * | 1995-02-06 | 1997-07-15 | Molecular Probes, Inc. | Methods of sensing with fluorescent conjugates of metal-chelating nitrogen heterocycles |
US20060014929A9 (en) * | 1996-07-17 | 2006-01-19 | Abdel-Magid Ahmed F | Liquid phase peptide synthesis of KL-4 pulmonary surfactant |
US6013764A (en) * | 1996-07-17 | 2000-01-11 | Ortho Pharmaceutical Corp. | Liquid phase peptide synthesis of KL-4 pulmonary surfactant |
US6492490B1 (en) * | 1996-07-17 | 2002-12-10 | Ortho Pharmaceutical Corporation | Liquid phase peptide syntheses of KL-4 pulmonary surfactant |
US20070149765A1 (en) * | 1996-07-17 | 2007-06-28 | Abdel-Magid Ahmed F | Liquid phase peptide syntheses of KL-4 pulmonary surfactant |
US20090259021A1 (en) * | 1996-07-17 | 2009-10-15 | Abdel-Magid Ahmed F | Liquid Phase Peptide Synthesis of KL-4 Pulmonary Surfactant |
US20020147302A1 (en) * | 1996-07-17 | 2002-10-10 | Abdel-Magid Ahmed F. | Liquid phase peptide synthesis of KL-4 pulmonary surfactant |
US8217142B2 (en) | 1996-07-17 | 2012-07-10 | Ortho Pharmaceutical Corporation | Liquid phase peptide synthesis of KL-4 pulmonary surfactant |
US5747345A (en) * | 1996-08-15 | 1998-05-05 | Johnson & Johnson Clinical Diagnostics, Inc. | Calcium specific diaza-18-crown-6-ether chromoionophores |
US6670449B1 (en) | 1997-02-13 | 2003-12-30 | Memorial Sloan-Kettering Cancer Center | Hybrid molecules and their use for optically detecting changes in cellular microenvironments |
US6180411B1 (en) | 1997-08-01 | 2001-01-30 | The Regents Of The University Of California | Light-triggered indicators that memorize analyte concentrations |
US8439872B2 (en) | 1998-03-30 | 2013-05-14 | Sanofi-Aventis Deutschland Gmbh | Apparatus and method for penetration with shaft having a sensor for sensing penetration depth |
US6171866B1 (en) | 1998-09-30 | 2001-01-09 | Avl Medical Instruments | Luminescence indicator for determining calcium ions |
DE19915137C2 (en) * | 1999-03-26 | 2001-10-18 | Michael Schaefer | Fluorescence microscopy method for quantifying multiple fluorochromes in a multi-colored sample and uses of the method |
US7060793B2 (en) | 1999-05-21 | 2006-06-13 | The Regents Of The University Of California | Circularly permuted fluorescent protein indicators |
US6469154B1 (en) | 1999-05-21 | 2002-10-22 | The Regents Of The University Of California | Fluorescent protein indicators |
US6699687B1 (en) | 1999-05-21 | 2004-03-02 | The Regents Of The University Of California | Circularly permuted fluorescent protein indicators |
US20020157120A1 (en) * | 1999-05-21 | 2002-10-24 | Tsien Roger Y. | Circularly permuted fluorescent protein indicators |
US20040147747A1 (en) * | 2000-08-04 | 2004-07-29 | Molecular Probes, Inc. | Derivatives of 1,2-dihydro-7-hydroxyquinolines containing fused rings |
US7816519B2 (en) | 2000-08-04 | 2010-10-19 | Life Technologies Corporation | Derivatives of 1,2-dihydro-7-hydroxyquinolines containing fused rings |
US8846925B2 (en) | 2000-08-04 | 2014-09-30 | Life Technologies Corporation | Derivatives of 1,2-dihydro-7-hydroxyquinolines containing fused rings |
US20110027800A1 (en) * | 2000-08-04 | 2011-02-03 | Life Technologies Corporation | Derivatives of 1,2-dihydro-7-hydroxyquinolines Containing Fused Rings |
US7169922B2 (en) | 2000-08-04 | 2007-01-30 | Invitrogen Corporation | Derivatives of 1,2-dihydro-7-hydroxyquinolines containing fused rings |
US20070093652A1 (en) * | 2000-08-04 | 2007-04-26 | Invitrogen Corporation | Derivatives of 1,2-dihydro-7-hydroxyquinolines containing fused rings |
US8183375B2 (en) | 2000-08-04 | 2012-05-22 | Life Technologies Corporation | Derivatives of 1,2-dihydro-7-hydroxyquinolines containing fused rings |
US8641644B2 (en) | 2000-11-21 | 2014-02-04 | Sanofi-Aventis Deutschland Gmbh | Blood testing apparatus having a rotatable cartridge with multiple lancing elements and testing means |
US7579463B2 (en) | 2000-12-20 | 2009-08-25 | Life Technologies Corporation | Crown ether derivatives |
US6962992B2 (en) | 2000-12-20 | 2005-11-08 | Molecullar Probes, Inc. | Crown ether derivatives |
US7989617B2 (en) | 2000-12-20 | 2011-08-02 | Life Technologies Corporation | Crown ether derivatives |
US20070043212A1 (en) * | 2000-12-20 | 2007-02-22 | Molecular Probes, Inc. | Crown ether derivatives |
US7988645B2 (en) | 2001-06-12 | 2011-08-02 | Pelikan Technologies, Inc. | Self optimizing lancing device with adaptation means to temporal variations in cutaneous properties |
US7981055B2 (en) | 2001-06-12 | 2011-07-19 | Pelikan Technologies, Inc. | Tissue penetration device |
US8360991B2 (en) | 2001-06-12 | 2013-01-29 | Sanofi-Aventis Deutschland Gmbh | Tissue penetration device |
US8343075B2 (en) | 2001-06-12 | 2013-01-01 | Sanofi-Aventis Deutschland Gmbh | Tissue penetration device |
US8337421B2 (en) | 2001-06-12 | 2012-12-25 | Sanofi-Aventis Deutschland Gmbh | Tissue penetration device |
US9802007B2 (en) | 2001-06-12 | 2017-10-31 | Sanofi-Aventis Deutschland Gmbh | Methods and apparatus for lancet actuation |
US8382683B2 (en) | 2001-06-12 | 2013-02-26 | Sanofi-Aventis Deutschland Gmbh | Tissue penetration device |
US8845550B2 (en) | 2001-06-12 | 2014-09-30 | Sanofi-Aventis Deutschland Gmbh | Tissue penetration device |
US9694144B2 (en) | 2001-06-12 | 2017-07-04 | Sanofi-Aventis Deutschland Gmbh | Sampling module device and method |
US9937298B2 (en) | 2001-06-12 | 2018-04-10 | Sanofi-Aventis Deutschland Gmbh | Tissue penetration device |
US8282577B2 (en) | 2001-06-12 | 2012-10-09 | Sanofi-Aventis Deutschland Gmbh | Method and apparatus for lancet launching device integrated onto a blood-sampling cartridge |
US8721671B2 (en) | 2001-06-12 | 2014-05-13 | Sanofi-Aventis Deutschland Gmbh | Electric lancet actuator |
US8216154B2 (en) | 2001-06-12 | 2012-07-10 | Sanofi-Aventis Deutschland Gmbh | Tissue penetration device |
US9427532B2 (en) | 2001-06-12 | 2016-08-30 | Sanofi-Aventis Deutschland Gmbh | Tissue penetration device |
US8211037B2 (en) | 2001-06-12 | 2012-07-03 | Pelikan Technologies, Inc. | Tissue penetration device |
US8679033B2 (en) | 2001-06-12 | 2014-03-25 | Sanofi-Aventis Deutschland Gmbh | Tissue penetration device |
US8206317B2 (en) | 2001-06-12 | 2012-06-26 | Sanofi-Aventis Deutschland Gmbh | Tissue penetration device |
US8206319B2 (en) | 2001-06-12 | 2012-06-26 | Sanofi-Aventis Deutschland Gmbh | Tissue penetration device |
US8641643B2 (en) | 2001-06-12 | 2014-02-04 | Sanofi-Aventis Deutschland Gmbh | Sampling module device and method |
US8162853B2 (en) | 2001-06-12 | 2012-04-24 | Pelikan Technologies, Inc. | Tissue penetration device |
US8123700B2 (en) | 2001-06-12 | 2012-02-28 | Pelikan Technologies, Inc. | Method and apparatus for lancet launching device integrated onto a blood-sampling cartridge |
US8016774B2 (en) | 2001-06-12 | 2011-09-13 | Pelikan Technologies, Inc. | Tissue penetration device |
US8622930B2 (en) | 2001-06-12 | 2014-01-07 | Sanofi-Aventis Deutschland Gmbh | Tissue penetration device |
US7909775B2 (en) | 2001-06-12 | 2011-03-22 | Pelikan Technologies, Inc. | Method and apparatus for lancet launching device integrated onto a blood-sampling cartridge |
EP2113773A2 (en) | 2001-10-12 | 2009-11-04 | Molecular Probes, Inc. | Antibody complexes and methods for immunolabeling |
WO2003030817A2 (en) | 2001-10-12 | 2003-04-17 | Molecular Probes, Inc. | Antibody complexes and methods for immunolabeling |
EP2259067A2 (en) | 2001-10-12 | 2010-12-08 | Molecular Probes, Inc. | Methods for immunolabeling |
US20070269902A1 (en) * | 2001-10-12 | 2007-11-22 | Joseph Beechem | Antibody complexes and methods for immunolabeling |
US20030073149A1 (en) * | 2001-10-12 | 2003-04-17 | Archer Robert A. | Antibody complexes and methods for immunolabeling |
US8323903B2 (en) | 2001-10-12 | 2012-12-04 | Life Technologies Corporation | Antibody complexes and methods for immunolabeling |
US9560993B2 (en) | 2001-11-21 | 2017-02-07 | Sanofi-Aventis Deutschland Gmbh | Blood testing apparatus having a rotatable cartridge with multiple lancing elements and testing means |
US20050181464A1 (en) * | 2002-04-04 | 2005-08-18 | Affinium Pharmaceuticals, Inc. | Novel purified polypeptides from bacteria |
US8157748B2 (en) | 2002-04-19 | 2012-04-17 | Pelikan Technologies, Inc. | Methods and apparatus for lancet actuation |
US8562545B2 (en) | 2002-04-19 | 2013-10-22 | Sanofi-Aventis Deutschland Gmbh | Tissue penetration device |
US9089294B2 (en) | 2002-04-19 | 2015-07-28 | Sanofi-Aventis Deutschland Gmbh | Analyte measurement device with a single shot actuator |
US8366637B2 (en) | 2002-04-19 | 2013-02-05 | Sanofi-Aventis Deutschland Gmbh | Method and apparatus for penetrating tissue |
US9072842B2 (en) | 2002-04-19 | 2015-07-07 | Sanofi-Aventis Deutschland Gmbh | Method and apparatus for penetrating tissue |
US8360992B2 (en) | 2002-04-19 | 2013-01-29 | Sanofi-Aventis Deutschland Gmbh | Method and apparatus for penetrating tissue |
US8337420B2 (en) | 2002-04-19 | 2012-12-25 | Sanofi-Aventis Deutschland Gmbh | Tissue penetration device |
US8337419B2 (en) | 2002-04-19 | 2012-12-25 | Sanofi-Aventis Deutschland Gmbh | Tissue penetration device |
US8905945B2 (en) | 2002-04-19 | 2014-12-09 | Dominique M. Freeman | Method and apparatus for penetrating tissue |
US9839386B2 (en) | 2002-04-19 | 2017-12-12 | Sanofi-Aventis Deustschland Gmbh | Body fluid sampling device with capacitive sensor |
US8333710B2 (en) | 2002-04-19 | 2012-12-18 | Sanofi-Aventis Deutschland Gmbh | Tissue penetration device |
US8845549B2 (en) | 2002-04-19 | 2014-09-30 | Sanofi-Aventis Deutschland Gmbh | Method for penetrating tissue |
US9724021B2 (en) | 2002-04-19 | 2017-08-08 | Sanofi-Aventis Deutschland Gmbh | Method and apparatus for penetrating tissue |
US8808201B2 (en) | 2002-04-19 | 2014-08-19 | Sanofi-Aventis Deutschland Gmbh | Methods and apparatus for penetrating tissue |
US8784335B2 (en) | 2002-04-19 | 2014-07-22 | Sanofi-Aventis Deutschland Gmbh | Body fluid sampling device with a capacitive sensor |
US9089678B2 (en) | 2002-04-19 | 2015-07-28 | Sanofi-Aventis Deutschland Gmbh | Method and apparatus for penetrating tissue |
US8382682B2 (en) | 2002-04-19 | 2013-02-26 | Sanofi-Aventis Deutschland Gmbh | Method and apparatus for penetrating tissue |
US8690796B2 (en) | 2002-04-19 | 2014-04-08 | Sanofi-Aventis Deutschland Gmbh | Method and apparatus for penetrating tissue |
US8388551B2 (en) | 2002-04-19 | 2013-03-05 | Sanofi-Aventis Deutschland Gmbh | Method and apparatus for multi-use body fluid sampling device with sterility barrier release |
US8403864B2 (en) | 2002-04-19 | 2013-03-26 | Sanofi-Aventis Deutschland Gmbh | Method and apparatus for penetrating tissue |
US8267870B2 (en) | 2002-04-19 | 2012-09-18 | Sanofi-Aventis Deutschland Gmbh | Method and apparatus for body fluid sampling with hybrid actuation |
US8235915B2 (en) | 2002-04-19 | 2012-08-07 | Sanofi-Aventis Deutschland Gmbh | Method and apparatus for penetrating tissue |
US8221334B2 (en) | 2002-04-19 | 2012-07-17 | Sanofi-Aventis Deutschland Gmbh | Method and apparatus for penetrating tissue |
US8636673B2 (en) | 2002-04-19 | 2014-01-28 | Sanofi-Aventis Deutschland Gmbh | Tissue penetration device |
US7875047B2 (en) | 2002-04-19 | 2011-01-25 | Pelikan Technologies, Inc. | Method and apparatus for a multi-use body fluid sampling device with sterility barrier release |
US9498160B2 (en) | 2002-04-19 | 2016-11-22 | Sanofi-Aventis Deutschland Gmbh | Method for penetrating tissue |
US7892183B2 (en) | 2002-04-19 | 2011-02-22 | Pelikan Technologies, Inc. | Method and apparatus for body fluid sampling and analyte sensing |
US9186468B2 (en) | 2002-04-19 | 2015-11-17 | Sanofi-Aventis Deutschland Gmbh | Method and apparatus for penetrating tissue |
US7901365B2 (en) | 2002-04-19 | 2011-03-08 | Pelikan Technologies, Inc. | Method and apparatus for penetrating tissue |
US7909778B2 (en) | 2002-04-19 | 2011-03-22 | Pelikan Technologies, Inc. | Method and apparatus for penetrating tissue |
US7909777B2 (en) | 2002-04-19 | 2011-03-22 | Pelikan Technologies, Inc | Method and apparatus for penetrating tissue |
US7909774B2 (en) | 2002-04-19 | 2011-03-22 | Pelikan Technologies, Inc. | Method and apparatus for penetrating tissue |
US9339612B2 (en) | 2002-04-19 | 2016-05-17 | Sanofi-Aventis Deutschland Gmbh | Tissue penetration device |
US7914465B2 (en) | 2002-04-19 | 2011-03-29 | Pelikan Technologies, Inc. | Method and apparatus for penetrating tissue |
US8579831B2 (en) | 2002-04-19 | 2013-11-12 | Sanofi-Aventis Deutschland Gmbh | Method and apparatus for penetrating tissue |
US9314194B2 (en) | 2002-04-19 | 2016-04-19 | Sanofi-Aventis Deutschland Gmbh | Tissue penetration device |
US7938787B2 (en) | 2002-04-19 | 2011-05-10 | Pelikan Technologies, Inc. | Method and apparatus for penetrating tissue |
US8202231B2 (en) | 2002-04-19 | 2012-06-19 | Sanofi-Aventis Deutschland Gmbh | Method and apparatus for penetrating tissue |
US8574168B2 (en) | 2002-04-19 | 2013-11-05 | Sanofi-Aventis Deutschland Gmbh | Method and apparatus for a multi-use body fluid sampling device with analyte sensing |
US7959582B2 (en) | 2002-04-19 | 2011-06-14 | Pelikan Technologies, Inc. | Method and apparatus for penetrating tissue |
US8197423B2 (en) | 2002-04-19 | 2012-06-12 | Pelikan Technologies, Inc. | Method and apparatus for penetrating tissue |
US7976476B2 (en) | 2002-04-19 | 2011-07-12 | Pelikan Technologies, Inc. | Device and method for variable speed lancet |
US8197421B2 (en) | 2002-04-19 | 2012-06-12 | Pelikan Technologies, Inc. | Method and apparatus for penetrating tissue |
US7981056B2 (en) | 2002-04-19 | 2011-07-19 | Pelikan Technologies, Inc. | Methods and apparatus for lancet actuation |
US8372016B2 (en) | 2002-04-19 | 2013-02-12 | Sanofi-Aventis Deutschland Gmbh | Method and apparatus for body fluid sampling and analyte sensing |
US8414503B2 (en) | 2002-04-19 | 2013-04-09 | Sanofi-Aventis Deutschland Gmbh | Methods and apparatus for lancet actuation |
US7988644B2 (en) | 2002-04-19 | 2011-08-02 | Pelikan Technologies, Inc. | Method and apparatus for a multi-use body fluid sampling device with sterility barrier release |
US9907502B2 (en) | 2002-04-19 | 2018-03-06 | Sanofi-Aventis Deutschland Gmbh | Method and apparatus for penetrating tissue |
US8007446B2 (en) | 2002-04-19 | 2011-08-30 | Pelikan Technologies, Inc. | Method and apparatus for penetrating tissue |
US8556829B2 (en) | 2002-04-19 | 2013-10-15 | Sanofi-Aventis Deutschland Gmbh | Method and apparatus for penetrating tissue |
US8496601B2 (en) | 2002-04-19 | 2013-07-30 | Sanofi-Aventis Deutschland Gmbh | Methods and apparatus for lancet actuation |
US8491500B2 (en) | 2002-04-19 | 2013-07-23 | Sanofi-Aventis Deutschland Gmbh | Methods and apparatus for lancet actuation |
US9226699B2 (en) | 2002-04-19 | 2016-01-05 | Sanofi-Aventis Deutschland Gmbh | Body fluid sampling module with a continuous compression tissue interface surface |
US9795334B2 (en) | 2002-04-19 | 2017-10-24 | Sanofi-Aventis Deutschland Gmbh | Method and apparatus for penetrating tissue |
US8062231B2 (en) | 2002-04-19 | 2011-11-22 | Pelikan Technologies, Inc. | Method and apparatus for penetrating tissue |
US9248267B2 (en) | 2002-04-19 | 2016-02-02 | Sanofi-Aventis Deustchland Gmbh | Tissue penetration device |
US8079960B2 (en) | 2002-04-19 | 2011-12-20 | Pelikan Technologies, Inc. | Methods and apparatus for lancet actuation |
US8430828B2 (en) | 2002-04-19 | 2013-04-30 | Sanofi-Aventis Deutschland Gmbh | Method and apparatus for a multi-use body fluid sampling device with sterility barrier release |
US8435190B2 (en) | 2002-04-19 | 2013-05-07 | Sanofi-Aventis Deutschland Gmbh | Method and apparatus for penetrating tissue |
US7102005B2 (en) | 2002-05-03 | 2006-09-05 | Molecular Probes, Inc. | Compositions and methods for detection and isolation of phosphorylated molecules |
US20070054304A1 (en) * | 2002-05-03 | 2007-03-08 | Invitrogen Corporation | Compositions and methods for detection and isolation of phosphorylated molecules |
US7776533B2 (en) | 2002-05-03 | 2010-08-17 | Life Technologies Corporation | Compositions and methods for detection and isolation of phosphorylated molecules |
US20040171034A1 (en) * | 2002-05-03 | 2004-09-02 | Brian Agnew | Compositions and methods for detection and isolation of phosphorylated molecules |
US7445894B2 (en) | 2002-05-03 | 2008-11-04 | Molecular Probes, Inc. | Compositions and methods for detection and isolation of phosphorylated molecules |
US20040038306A1 (en) * | 2002-05-03 | 2004-02-26 | Brian Agnew | Compositions and methods for detection and isolation of phosphorylated molecules |
US20050014197A1 (en) * | 2002-05-03 | 2005-01-20 | Brian Agnew | Compositions and methods for detection and isolation of phosphorylated molecules |
US20090081722A1 (en) * | 2002-09-12 | 2009-03-26 | Invitrogen Corporation | Site-specific labeling of affinity tags in fusion proteins |
US9164099B2 (en) | 2002-09-12 | 2015-10-20 | Life Technologies Corporation | Site-specific labeling of affinity tags in fusion proteins |
US7465848B2 (en) | 2002-11-20 | 2008-12-16 | The General Hospital Corporation | Zebrafish assay |
US8574895B2 (en) | 2002-12-30 | 2013-11-05 | Sanofi-Aventis Deutschland Gmbh | Method and apparatus using optical techniques to measure analyte levels |
US9034639B2 (en) | 2002-12-30 | 2015-05-19 | Sanofi-Aventis Deutschland Gmbh | Method and apparatus using optical techniques to measure analyte levels |
US8445702B2 (en) | 2003-05-05 | 2013-05-21 | Life Technologies Corporation | Zinc binding compounds and their method of use |
US20110159517A1 (en) * | 2003-05-05 | 2011-06-30 | Life Technologies Corporation | Zinc binding compounds and their method of use |
US8262614B2 (en) | 2003-05-30 | 2012-09-11 | Pelikan Technologies, Inc. | Method and apparatus for fluid injection |
US8251921B2 (en) | 2003-06-06 | 2012-08-28 | Sanofi-Aventis Deutschland Gmbh | Method and apparatus for body fluid sampling and analyte sensing |
US9144401B2 (en) | 2003-06-11 | 2015-09-29 | Sanofi-Aventis Deutschland Gmbh | Low pain penetrating member |
US10034628B2 (en) | 2003-06-11 | 2018-07-31 | Sanofi-Aventis Deutschland Gmbh | Low pain penetrating member |
US7582461B2 (en) | 2003-07-29 | 2009-09-01 | Life Technologies Corporation | Kinase and phosphatase assays |
US20100240080A1 (en) * | 2003-07-29 | 2010-09-23 | Life Technologies Corporation | Kinase and phosphatase assays |
US7619059B2 (en) | 2003-07-29 | 2009-11-17 | Life Technologies Corporation | Bimolecular optical probes |
US8067536B2 (en) | 2003-07-29 | 2011-11-29 | Life Technologies Corporation | Kinase and phosphatase assays |
US7727752B2 (en) | 2003-07-29 | 2010-06-01 | Life Technologies Corporation | Kinase and phosphatase assays |
US20110105362A1 (en) * | 2003-08-11 | 2011-05-05 | Life Technologies Corporation | Cyanine compounds and their application as quenching compounds |
US9150922B2 (en) | 2003-08-11 | 2015-10-06 | Life Technologies Corporation | Cyanine compounds and their application as quenching compounds |
US20050191643A1 (en) * | 2003-08-11 | 2005-09-01 | Rosaria Haugland | Cyanine compounds and their application as quenching compounds |
US7271265B2 (en) | 2003-08-11 | 2007-09-18 | Invitrogen Corporation | Cyanine compounds and their application as quenching compounds |
US20080039630A1 (en) * | 2003-08-11 | 2008-02-14 | Invitrogen Corporation | Cyanine compounds and their application as quenching compounds |
US20050064485A1 (en) * | 2003-09-12 | 2005-03-24 | Kurt Vogel | Multiplex binding and activity assays |
US20060160068A1 (en) * | 2003-09-17 | 2006-07-20 | Joseph Beechem | Competitive immunoassay |
US7282339B2 (en) | 2003-09-17 | 2007-10-16 | Invitrogen Corporation | Competitive immunoassay |
US8945910B2 (en) | 2003-09-29 | 2015-02-03 | Sanofi-Aventis Deutschland Gmbh | Method and apparatus for an improved sample capture device |
US8282576B2 (en) | 2003-09-29 | 2012-10-09 | Sanofi-Aventis Deutschland Gmbh | Method and apparatus for an improved sample capture device |
US9351680B2 (en) | 2003-10-14 | 2016-05-31 | Sanofi-Aventis Deutschland Gmbh | Method and apparatus for a variable user interface |
US20080160627A1 (en) * | 2003-11-19 | 2008-07-03 | Invitrogen Corporation | Environmental Sensitive Fluorogenic Compounds and Their Application for Singlet Oxygen and Protein Detection |
US8173698B2 (en) | 2003-11-19 | 2012-05-08 | Life Technologies Corporation | Environmental sensitive fluorogenic compounds and their application for singlet oxygen and protein detection |
US9403985B2 (en) | 2003-12-05 | 2016-08-02 | Life Technologies Corporation | Methine-substituted cyanine dye compounds |
US20050208534A1 (en) * | 2003-12-05 | 2005-09-22 | Dallwig Jason A | Methine-substituted cyanine dye compounds |
US10005908B2 (en) | 2003-12-05 | 2018-06-26 | Life Technologies Corporation | Methine-substituted cyanine dye compounds |
US7446202B2 (en) | 2003-12-05 | 2008-11-04 | Molecular Probes, Inc. | Cyanine dye compounds |
US9040561B2 (en) | 2003-12-05 | 2015-05-26 | Life Technologies Corporation | Methine-substituted cyanine dye compounds |
US7842811B2 (en) | 2003-12-05 | 2010-11-30 | Life Technologies Corporation | Cyanine dye compounds |
US7655409B2 (en) | 2003-12-05 | 2010-02-02 | Life Technologies Corporation | Cyanine dye compounds |
US7776529B2 (en) | 2003-12-05 | 2010-08-17 | Life Technologies Corporation | Methine-substituted cyanine dye compounds |
US20100317543A1 (en) * | 2003-12-05 | 2010-12-16 | Life Technologies Corporation | Methine-substituted cyanine dye compounds |
US8470529B2 (en) | 2003-12-05 | 2013-06-25 | Life Technologies Corporation | Methine-substituted cyanine dye compounds |
US20100120051A1 (en) * | 2003-12-05 | 2010-05-13 | Life Technologies Corporation | Cyanine dye compounds |
US20090047683A1 (en) * | 2003-12-05 | 2009-02-19 | Invitrogen Corporation | Cyanine dye compounds |
US8668656B2 (en) | 2003-12-31 | 2014-03-11 | Sanofi-Aventis Deutschland Gmbh | Method and apparatus for improving fluidic flow and sample capture |
US9561000B2 (en) | 2003-12-31 | 2017-02-07 | Sanofi-Aventis Deutschland Gmbh | Method and apparatus for improving fluidic flow and sample capture |
US8296918B2 (en) | 2003-12-31 | 2012-10-30 | Sanofi-Aventis Deutschland Gmbh | Method of manufacturing a fluid sampling device with improved analyte detecting member configuration |
US8865891B2 (en) | 2004-01-21 | 2014-10-21 | Life Technologies Corporation | Optically-detectable enzyme substrates and their method of use |
US8318450B2 (en) | 2004-01-21 | 2012-11-27 | Life Technologies Corporation | Optically-detectable enzyme substrates and their method of use |
US20050233467A1 (en) * | 2004-04-15 | 2005-10-20 | Akwasi Minta | Visible wavelength fluorescent calcium indicators that are (i) leakage resistant and (ii) operate near membranes |
US20050250214A1 (en) * | 2004-05-05 | 2005-11-10 | Gee Kyle R | Zinc binding compounds and their method of use |
US9261476B2 (en) | 2004-05-20 | 2016-02-16 | Sanofi Sa | Printable hydrogel for biosensors |
US8828203B2 (en) | 2004-05-20 | 2014-09-09 | Sanofi-Aventis Deutschland Gmbh | Printable hydrogels for biosensors |
US9820684B2 (en) | 2004-06-03 | 2017-11-21 | Sanofi-Aventis Deutschland Gmbh | Method and apparatus for a fluid sampling device |
US9775553B2 (en) | 2004-06-03 | 2017-10-03 | Sanofi-Aventis Deutschland Gmbh | Method and apparatus for a fluid sampling device |
WO2006023231A3 (en) * | 2004-07-27 | 2006-05-04 | Molecular Probes Inc | Fluorescent metal ion indicators with large stokes shift |
US9372181B2 (en) | 2004-07-27 | 2016-06-21 | Life Technologies Corporation | Fluorescent metal ion indicators with large stokes shifts |
US8785208B2 (en) | 2004-07-27 | 2014-07-22 | Life Technologies Corporation | Fluorescent metal ion indicators with large stokes shifts |
WO2006023231A2 (en) * | 2004-07-27 | 2006-03-02 | Molecular Probes, Inc. | Fluorescent metal ion indicators with large stokes shift |
US9846168B2 (en) | 2004-07-27 | 2017-12-19 | Life Technologies Corporation | Fluorescent metal ion indicators with large stokes shifts |
US8652831B2 (en) | 2004-12-30 | 2014-02-18 | Sanofi-Aventis Deutschland Gmbh | Method and apparatus for analyte measurement test time |
US9366676B2 (en) | 2005-05-11 | 2016-06-14 | Life Technologies Corporation | Fluorescent chemical compounds having high selectivity for double stranded DNA, and methods for their use |
US20100143917A1 (en) * | 2005-05-11 | 2010-06-10 | Life Technologies Corporation | Fluorescent chemical compounds having high selectivity for double stranded dna, and methods for their use |
US20110217699A1 (en) * | 2005-05-11 | 2011-09-08 | Life Technologies Corporation | Fluorescent Chemical Compounds Having High Selectivity for Double Stranded DNA, and Methods for Their Use |
US9115397B2 (en) | 2005-05-11 | 2015-08-25 | Life Technologies Corporation | Fluorescent chemical compounds having high selectivity for double stranded DNA, and methods for their use |
US7598390B2 (en) | 2005-05-11 | 2009-10-06 | Life Technologies Corporation | Fluorescent chemical compounds having high selectivity for double stranded DNA, and methods for their use |
US7943777B2 (en) | 2005-05-11 | 2011-05-17 | Life Technologies Corporation | Fluorescent chemical compounds having high selectivity for double stranded DNA, and methods for their use |
US8865904B2 (en) | 2005-05-11 | 2014-10-21 | Life Technologies Corporation | Fluorescent chemical compounds having high selectivity for double stranded DNA, and methods for their use |
DE102005022880A1 (en) * | 2005-05-18 | 2006-11-30 | Olympus Soft Imaging Solutions Gmbh | Separation of spectrally or color superimposed image contributions in a multi-color image, especially in transmission microscopic multi-color images |
US20090103801A1 (en) * | 2005-05-18 | 2009-04-23 | Olympus Soft Imaging Solutions Gmbh | Separation of Spectrally Overlaid or Color-Overlaid Image Contributions in a Multicolor Image, Especially Transmission Microscopic Multicolor Image |
DE102005022880B4 (en) * | 2005-05-18 | 2010-12-30 | Olympus Soft Imaging Solutions Gmbh | Separation of spectrally or color superimposed image contributions in a multi-color image, especially in transmission microscopic multi-color images |
US20100009342A1 (en) * | 2005-09-06 | 2010-01-14 | Life Technologies Corporation | Control of chemical modification |
US20070154958A1 (en) * | 2005-10-13 | 2007-07-05 | Aureon Laboratories, Inc. | Multiplex in situ immunohistochemical analysis |
US8093012B2 (en) | 2005-10-13 | 2012-01-10 | Aureon Laboratories, Inc. | Multiplex in situ immunohistochemical analysis |
US20070161112A1 (en) * | 2006-01-12 | 2007-07-12 | Invitrogen Corporation | Heavy metal binding compounds and their method of use |
US8680320B2 (en) | 2006-01-12 | 2014-03-25 | Life Technologies Corporation | Heavy metal binding compounds and their method of use |
US7521577B2 (en) | 2006-01-12 | 2009-04-21 | Molecular Probes, Inc. | Heavy metal binding compounds and their method of use |
US20100255601A1 (en) * | 2006-01-24 | 2010-10-07 | Invitrogen Corporation | Device and methods for quantifying analytes |
US10533946B2 (en) | 2006-01-24 | 2020-01-14 | Life Technologies Corporation | Device and methods for quantifying analytes |
US8551408B2 (en) | 2006-01-24 | 2013-10-08 | Life Technologies Corporation | Device and methods for quantifying analytes |
US11635383B2 (en) | 2006-01-24 | 2023-04-25 | Life Technologies Corporation | Device and methods for quantifying analytes introduction |
US8623282B2 (en) | 2006-01-24 | 2014-01-07 | Life Technologies Corporation | Device and methods for quantifying analytes |
US10962480B2 (en) | 2006-01-24 | 2021-03-30 | Life Technologies Corporation | Device and methods for quantifying analytes introduction |
US9964490B2 (en) | 2006-01-24 | 2018-05-08 | Life Technologies Corporation | Device and methods for quantifying analytes |
US20100167320A1 (en) * | 2006-01-30 | 2010-07-01 | Life Technologies Corporation | Compositions and Methods for Detecting and Quantifying Toxic Substances in Disease States |
EP2623610A2 (en) | 2006-02-10 | 2013-08-07 | Life Technologies Corporation | Labeling and detection of post translationally modified proteins |
EP3438131A1 (en) | 2006-02-10 | 2019-02-06 | Life Technologies Corporation | Oligosaccharide modification and labeling of proteins |
US10676771B2 (en) | 2006-02-10 | 2020-06-09 | Life Technologies Corporation | Oligosaccharide modification and labeling of proteins |
EP4092418A1 (en) | 2006-02-10 | 2022-11-23 | Life Technologies Corporation | Oligosaccharide modification and labeling of proteins |
EP2623986A2 (en) | 2006-02-10 | 2013-08-07 | Life Technologies Corporation | Labeling and detection of post translationally modified proteins |
EP2623609A2 (en) | 2006-02-10 | 2013-08-07 | Life Technologies Corporation | Labeling and detection of post translationally modified proteins |
US8785212B2 (en) | 2006-02-10 | 2014-07-22 | Life Technologies Corporation | Oligosaccharide modification and labeling of proteins |
US8716033B2 (en) | 2006-02-10 | 2014-05-06 | Life Technologies Corporation | Oligosaccharide modification and labeling of proteins |
US9645140B2 (en) | 2006-02-10 | 2017-05-09 | Life Technologies Corporation | Labeling and detection of post translationally modified proteins |
US20070190597A1 (en) * | 2006-02-10 | 2007-08-16 | Invitrogen Corporation | Oligosaccharide modification and labeling of proteins |
US20070249014A1 (en) * | 2006-02-10 | 2007-10-25 | Invitrogen Corporation | Labeling and detection of post translationally modified proteins |
US20110207171A1 (en) * | 2006-02-10 | 2011-08-25 | Life Technologies Corporation | Oligosaccharide modification and labeling of proteins |
US8562802B1 (en) | 2006-02-13 | 2013-10-22 | Life Technologies Corporation | Transilluminator base and scanner for imaging fluorescent gels, charging devices and portable electrophoresis systems |
EP2362223A2 (en) | 2006-03-23 | 2011-08-31 | Absorber AB | Blood group antigens of different types for diagnostic and therapeutic applications |
WO2007135571A2 (en) | 2006-03-23 | 2007-11-29 | Absorber Ab | Blood group antigens of different types for diagnostic and therapeutic applications |
EP2362224A2 (en) | 2006-03-23 | 2011-08-31 | Absorber AB | Blood group antigens of different types for diagnostic and therapeutic applications |
US8404456B2 (en) | 2006-03-23 | 2013-03-26 | Absorber Ab | Blood group antigens of different types for diagnostic and therapeutic applications |
US7897328B2 (en) | 2006-03-23 | 2011-03-01 | Absorber Ab | Blood group antigens of different types for diagnostic and therapeutic applications |
US20070224652A1 (en) * | 2006-03-23 | 2007-09-27 | Jan Holgersson | Blood Group Antigens of Different Types for Diagnostic and Therapeutic Applications |
WO2007124464A2 (en) | 2006-04-20 | 2007-11-01 | Becton, Dickinson And Company | Thermostable proteins and methods of making and using thereof |
US8124368B2 (en) | 2006-07-07 | 2012-02-28 | Life Technologies Corporation | Fluorogenic protein kinase substrates |
US20080009026A1 (en) * | 2006-07-07 | 2008-01-10 | Invitrogen Corporation | Fluorogenic protein kinase substrates |
US8702624B2 (en) | 2006-09-29 | 2014-04-22 | Sanofi-Aventis Deutschland Gmbh | Analyte measurement device with a single shot actuator |
EP2727965A1 (en) | 2006-10-27 | 2014-05-07 | Life Technologies Corporation | Fluorogenic pH sensitive dyes and their method of use |
US11867698B2 (en) | 2006-10-27 | 2024-01-09 | Life Technologies Corporation | Fluorogenic pH sensitive dyes and their method of use |
US9910051B2 (en) | 2006-10-27 | 2018-03-06 | Life Technologies Corporation | Fluorogenic pH sensitive dyes and their method of use |
US10845373B2 (en) | 2006-10-27 | 2020-11-24 | Life Technologies Corporation | Fluorogenic pH sensitive dyes and their method of use |
US9140706B2 (en) | 2007-01-30 | 2015-09-22 | Life Technologies Corporation | Labeling reagents and methods of their use |
US20090004753A1 (en) * | 2007-01-30 | 2009-01-01 | Invitrogen Corporation | Labeling reagents and methods of their use |
US8586743B2 (en) | 2007-01-30 | 2013-11-19 | Life Technologies Corporation | Labeling reagents and methods of their use |
US9645153B2 (en) | 2007-01-30 | 2017-05-09 | Life Technologies Corporation | Labeling reagents and methods of their use |
EP2224013A1 (en) | 2007-02-13 | 2010-09-01 | Life Technologies Corporation | Methods for the determination of telomerase activity using click chemistry |
EP2535423A2 (en) | 2007-02-13 | 2012-12-19 | Life Technologies Corporation | Labeling and detection of nucleic acids |
US8779165B2 (en) | 2007-04-13 | 2014-07-15 | Aat Bioquest, Inc. | Fluorescent ion indicators and their applications |
US9346778B2 (en) | 2007-04-13 | 2016-05-24 | Aat Bioquest, Inc. | Carbofluorescein lactone ion indicators and their applications |
US20080254498A1 (en) * | 2007-04-13 | 2008-10-16 | Abd Bioquest, Inc. | Fluorescent ion indicators and their applications |
US9097730B2 (en) | 2007-04-13 | 2015-08-04 | Aat Bioquest, Inc. | Fluorescein lactone ion indicators and their applications |
US20110229924A1 (en) * | 2007-04-13 | 2011-09-22 | Zhenjun Diwu | Fluorescent ion indicators and their applications |
US8927224B2 (en) | 2007-04-13 | 2015-01-06 | Aat Bioquest, Inc. | Fluorescent ion indicators and their applications |
US9279817B2 (en) | 2007-04-13 | 2016-03-08 | Aat Bioquest, Inc. | Carbofluorescein lactone ion indicators and their applications |
US8513451B2 (en) | 2007-05-30 | 2013-08-20 | Hee Chol Kang | Fluorescent phospholipase A2 indicators |
US20100203564A1 (en) * | 2007-05-30 | 2010-08-12 | Life Technologies Corporation | Fluorescent Phospholipase A2 Indicators |
US8304198B2 (en) | 2007-06-19 | 2012-11-06 | Rajiv Gandhi Centre For Biotechnology | Assay for detection of transient intracellular CA2+ |
US20100240071A1 (en) * | 2007-06-19 | 2010-09-23 | Rajiv Gandhi Centre For Biotechnology | Assay for Detection of Transient Intracellular CA2+ |
US9958462B2 (en) | 2007-06-19 | 2018-05-01 | Rajiv Gandhi Centre For Biotechnology | Assay for detection of transient intracellular Ca2+ |
KR100976230B1 (en) | 2007-09-07 | 2010-08-17 | 고려대학교 산학협력단 | Two photon probe for real time monitoring of intracellular calcium ions, method for preparing the same and method for real time monitoring of intracellular calcium ions |
WO2009031734A1 (en) * | 2007-09-07 | 2009-03-12 | Korea University Industrial & Academic Collaboration Foundation | Two-photon probe for real-time monitoring of intracellular calcium ions, method for preparing the probe and method for real-time monitoring of intracellular calcium ions using the probe |
US9386944B2 (en) | 2008-04-11 | 2016-07-12 | Sanofi-Aventis Deutschland Gmbh | Method and apparatus for analyte detecting device |
US10138510B2 (en) | 2008-05-16 | 2018-11-27 | Life Technologies Corporation | Dual labeling methods for measuring cellular proliferation |
US20110118142A1 (en) * | 2008-05-16 | 2011-05-19 | Life Technologies Corporation | Dual labeling methods for measuring cellular proliferation |
US8540521B2 (en) | 2008-12-29 | 2013-09-24 | Life Technologies Corporation | Benzoxazole-based fluorescent metal ion indicators |
US8968997B2 (en) | 2008-12-29 | 2015-03-03 | Life Technologies Corporation | Benzoxazole-based fluorescent metal ion indicators |
US9375169B2 (en) | 2009-01-30 | 2016-06-28 | Sanofi-Aventis Deutschland Gmbh | Cam drive for managing disposable penetrating member actions with a single motor and motor and control system |
WO2010099137A2 (en) | 2009-02-26 | 2010-09-02 | Osi Pharmaceuticals, Inc. | In situ methods for monitoring the emt status of tumor cells in vivo |
WO2011047391A1 (en) | 2009-10-17 | 2011-04-21 | Asante Research, Llc | Fluorescent calcium indicators that are ratiometric and emit in the red spectrum |
US8965476B2 (en) | 2010-04-16 | 2015-02-24 | Sanofi-Aventis Deutschland Gmbh | Tissue penetration device |
US8974651B2 (en) | 2010-04-17 | 2015-03-10 | C.C. Imex | Illuminator for visualization of fluorophores |
US9795747B2 (en) | 2010-06-02 | 2017-10-24 | Sanofi-Aventis Deutschland Gmbh | Methods and apparatus for lancet actuation |
WO2012022734A2 (en) | 2010-08-16 | 2012-02-23 | Medimmune Limited | Anti-icam-1 antibodies and methods of use |
WO2014066733A2 (en) | 2012-10-25 | 2014-05-01 | Life Technologies Corporation | Methods and compositions for enzyme-mediated site-specific radiolabeling of glycoproteins |
WO2014085160A1 (en) | 2012-11-29 | 2014-06-05 | Idexx Laboratories, Inc. | Multi-layer device for selectively determining magnesium ion |
US9360474B2 (en) | 2012-11-29 | 2016-06-07 | Opti Medical Systems, Inc. | Multi-layer device for selectively determining magnesium ion |
WO2015050959A1 (en) | 2013-10-01 | 2015-04-09 | Yale University | Anti-kit antibodies and methods of use thereof |
EP3733244A1 (en) | 2013-10-02 | 2020-11-04 | Medlmmune, LLC | Neutralizing anti-influenza a antibodies and uses thereof |
US9504405B2 (en) | 2013-10-23 | 2016-11-29 | Verily Life Sciences Llc | Spatial modulation of magnetic particles in vasculature by external magnetic field |
US9636034B2 (en) | 2013-10-23 | 2017-05-02 | Verily Life Sciences Llc | Non-invasive analyte detection system with modulation source |
US11464429B2 (en) | 2013-10-23 | 2022-10-11 | Verily Life Sciences Llc | Modulation of a response signal to distinguish between analyte and background signals |
US10542918B2 (en) | 2013-10-23 | 2020-01-28 | Verily Life Sciences Llc | Modulation of a response signal to distinguish between analyte and background signals |
US10641731B2 (en) | 2014-04-01 | 2020-05-05 | C.C. Imex | Electrophoresis running tank assembly |
US9835587B2 (en) | 2014-04-01 | 2017-12-05 | C.C. Imex | Electrophoresis running tank assembly |
WO2016111915A1 (en) | 2015-01-06 | 2016-07-14 | De Haas Anthony H | Near-infrared fluorescent surgical dye markers |
US10383957B2 (en) | 2015-01-06 | 2019-08-20 | Anthony H. de Haas | Near-infrared fluorescent surgical dye markers |
US9861710B1 (en) | 2015-01-16 | 2018-01-09 | Verily Life Sciences Llc | Composite particles, methods, and in vivo diagnostic system |
US10028659B2 (en) | 2015-03-26 | 2018-07-24 | Verily Life Sciences Llc | Aptamer-based sensors, implantable devices and detection system |
WO2017181176A1 (en) | 2016-04-15 | 2017-10-19 | Lcahn School Of Medicine At Mount Sinai | Tissue profiling using multiplexed immunohistochemical consecutive staining |
WO2018183621A1 (en) | 2017-03-30 | 2018-10-04 | Life Technologies Corporation | Quantification of ngs dna by adapter sequence |
US9810700B1 (en) | 2017-05-31 | 2017-11-07 | Aat Bioquest, Inc. | Fluorogenic calcium ion indicators and methods of using the same |
CN111056985A (en) * | 2020-01-02 | 2020-04-24 | 广州大学 | Partially cyanine derivative fluorescent probe and preparation method and application thereof |
CN111056985B (en) * | 2020-01-02 | 2022-12-27 | 广州大学 | Partially cyanine derivative fluorescent probe and preparation method and application thereof |
CN111138387A (en) * | 2020-01-02 | 2020-05-12 | 广州大学 | Cyano furanone derivative fluorescent probe and preparation method and application thereof |
WO2022006368A2 (en) | 2020-07-02 | 2022-01-06 | Life Technologies Corporation | Trinucleotide cap analogs, preparation and uses thereof |
WO2023004400A1 (en) | 2021-07-21 | 2023-01-26 | Life Technologies Corporation | Dibenzoxanthene quenchers, uses, and methods of preparation |
Also Published As
Publication number | Publication date |
---|---|
JPS61100599A (en) | 1986-05-19 |
DE3566112D1 (en) | 1988-12-15 |
CA1254896A (en) | 1989-05-30 |
EP0178775B1 (en) | 1988-11-09 |
JPH0694444B2 (en) | 1994-11-24 |
EP0178775A2 (en) | 1986-04-23 |
ATE38509T1 (en) | 1988-11-15 |
EP0178775A3 (en) | 1986-08-27 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
US4603209A (en) | Fluorescent indicator dyes for calcium ions | |
Grynkiewicz et al. | A new generation of Ca2+ indicators with greatly improved fluorescence properties. | |
US5516911A (en) | Fluorescent intracellular calcium indicators | |
US4849362A (en) | Fluorescent intracellular calcium indicators | |
US4945171A (en) | Xanthene dyes having a fused (C) benzo ring | |
US5049673A (en) | Fluorescent indicator dyes for calcium working at long wavelengths | |
US4774339A (en) | Chemically reactive dipyrrometheneboron difluoride dyes | |
JP3224419B2 (en) | Merocyanine and nitro- or nitroso-substituted polyhalogenated phenolsulfonephthalein as protein indicators in biological samples | |
US4689432A (en) | Chelators whose affinity for calcium is decreased by illumination | |
US5409835A (en) | Long-wavelength fluorescent probe compounds for calcium ions and their use in ratiometrically measuring calcium ion concentrations | |
US5089630A (en) | Dioxetanes for use in assays | |
JPS588783A (en) | Fluorescent chelate | |
JP4065053B2 (en) | Method for quantifying alkali ions and monoaza-crown ether | |
US5310922A (en) | Squaraine dyes | |
JP2006219453A (en) | Metal-discrimination-type two-color fluorescent molecule containing quinoline ring as parent nucleus | |
EP0476456A2 (en) | Substituted 2-thiazolyl tetrazolium salt indicators | |
JP2000109452A (en) | Luminescent indicator for measuring calcium ion | |
US4677060A (en) | Process for determining the pH value in the interior of a cell | |
PT594047E (en) | CROMOIONOFOROS ARILAZO | |
JPH0655158B2 (en) | Fluorogenic substrate | |
JPH06503844A (en) | Substituted thiazoline-dioxetane substrates, methods for their preparation and uses thereof | |
Tsien et al. | Fluorescent indicator dyes for calcium ions | |
JP5906190B2 (en) | Fluorescent calcium indicator that is ratiometric and emits light in the red spectrum | |
US4608435A (en) | Benzo-1,4-quinones | |
Darcy et al. | Photochromic systems. Part 1. Photochemical studies on (E)-2-isopropylidene-3-[1-(3, 4, 5-trimethoxyphenyl) ethylidene] succinic anhydride and related compounds |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
AS | Assignment |
Owner name: REGENTS OF THE UNIVERSITY OF CALIFORNIA, THE, 219 Free format text: ASSIGNMENT OF ASSIGNORS INTEREST.;ASSIGNOR:TSIEN, ROGER Y.;REEL/FRAME:004337/0161 Effective date: 19840917 Owner name: REGENTS OF THE UNIVERSITY OF CALIFORNIA, THE, 2199 Free format text: ASSIGNMENT OF ASSIGNORS INTEREST.;ASSIGNOR:GRYNKIEWICZ, GRZGORZ;REEL/FRAME:004337/0162 Effective date: 19840914 Owner name: REGENTS OF THE UNIVERSITY OF CALIFORNIA, THE,CALIF Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNOR:TSIEN, ROGER Y.;REEL/FRAME:004337/0161 Effective date: 19840917 Owner name: REGENTS OF THE UNIVERSITY OF CALIFORNIA, THE,CALIF Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNOR:GRYNKIEWICZ, GRZGORZ;REEL/FRAME:004337/0162 Effective date: 19840914 |
|
STCF | Information on status: patent grant |
Free format text: PATENTED CASE |
|
CC | Certificate of correction | ||
FEPP | Fee payment procedure |
Free format text: PAYOR NUMBER ASSIGNED (ORIGINAL EVENT CODE: ASPN); ENTITY STATUS OF PATENT OWNER: LARGE ENTITY Free format text: PAT HLDR NO LONGER CLAIMS SMALL ENT STAT AS NONPROFIT ORG (ORIGINAL EVENT CODE: LSM3); ENTITY STATUS OF PATENT OWNER: LARGE ENTITY |
|
FPAY | Fee payment |
Year of fee payment: 4 |
|
FPAY | Fee payment |
Year of fee payment: 8 |
|
FPAY | Fee payment |
Year of fee payment: 12 |